LOCUS       pCAG-3XFLAG-hRAD21-mCHE 7169 bp ds-DNA     circular     03-FEB-2025
DEFINITION  .
KEYWORDS    "accession:addgene_156448_301985"
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
COMMENT     Imported from database: AddGene Entry: pKS070 -
            pCAGGS-3XFLAG-(human)CTCF-eGFP URL:
            https://www.addgene.org/browse/sequence/301985/ GENERAL Depositing
            Lab: Elphege Nora Publication: Nora et al Nat Commun. 2020 Nov
            5;11(1):5612. doi: 10.1038/s41467-020-19283-x. Purpose: For
            transient expression of human CTCF tagged with eGFP BACKBONE Vector
            backbone: pKS070-pCAGGS-3XFLAG-(human)CTCF-eGFP Vector type:
            Mammalian Expression CLONING INFORMATION Cloning method: Gibson
            Cloning GROWTH IN BACTERIA Bacterial Resistance(s): Ampicillin Copy
            number: High Copy Growth Strain(s): DH5alpha Growth Temperature:
            37°C GENE/INSERT Entrez Gene:  Gene/Insert name: human CTCF
            Mutation: none Species: H. sapiens (human) TERMS AND LICENSES
            Academic/Nonprofit Terms:  Industry Terms:
FEATURES             Location/Qualifiers
     enhancer        4..383
                     /label="CMV enhancer"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
                     /note="note: human cytomegalovirus immediate early enhancer"
     primer          41..63
                     /label="Kostja-CMV-DsRed-PciI-f"
                     /note="sequence: tagtaaacatgtacggggtcattagttcatagcc"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     primer          complement(42..63)
                     /label="tlCMV_31"
                     /note="sequence: GGCTATGAACTAATGACCCCGT"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          complement(43..63)
                     /label="tgfbr_checkup_R"
                     /note="sequence: GGCTATGAACTAATGACCCCG"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     primer          complement(85..108)
                     /label="CMV enh 5R"
                     /note="sequence: gcgggccatttaccgtaagttatg"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     primer          complement(85..108)
                     /label="gib-TandemPolyA2 R"
                     /note="sequence: agcgcagcgagtcagtgagcgaggagcgggccatttaccgtaagttatg"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     primer          complement(93..113)
                     /label="tlCMV313"
                     /note="sequence: acgatggactccagTCcggccTGCCAGGCGGGCCATTTACCGT"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer          complement(95..113)
                     /label="TAIL-5'Cas9-1"
                     /note="sequence: AATGCCAGGCGGGCCATTTACC"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          complement(121..143)
                     /label="tlCMV314"
                     /note="sequence: CGTCAATGGGCGGGGGTCGTTGG"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer          126..145
                     /label="CMV1_F"
                     /note="sequence: GACCCCCGCCCATTGACGTC"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          complement(158..187)
                     /label="Kostja-gBlock4-r"
                     /note="sequence: AAAGTCCCTANTGGCGTTACTATGGGAACA"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     primer          complement(168..189)
                     /label="CDC7_up_check_R:"
                     /note="sequence: GGAAAGTCCCTATTGGCGTTAC"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer          172..190
                     /label="integProm_L"
                     /note="sequence: CGCCAATAGGGACTTTCCA"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     primer          177..206
                     /label="Kostja-CMV-f"
                     /note="sequence: ATAGGGACTTNCCATTGACGTCAATGGGTG"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     primer          complement(248..272)
                     /label="TAIL-5'Cas9-2enh"
                     /note="sequence: acgatggaactccagtccggccagggggcgtacttggcatatgatac"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer          complement(286..307)
                     /label="tlCMV313"
                     /note="sequence: acgatggactccagTCcggccTGCCAGGCGGGCCATTTACCGT"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer          288..307
                     /label="CMV511"
                     /note="sequence: GGTAAATGGCCCGCCTGGCA"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer          complement(288..309)
                     /label="TAIL-5'Cas9-1"
                     /note="sequence: AATGCCAGGCGGGCCATTTACC"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          299..320
                     /label="Primer4"
                     /note="sequence: CGCCTGGCATTATGCCCAGTAC"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     primer          complement(384..408)
                     /label="tlCMV312"
                     /note="sequence: GCAGAACGTGGGGCTCACCTCGACC"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     promoter        386..661
                     /label="chicken beta-actin promoter"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     primer          complement(388..408)
                     /label="CMV311"
                     /note="sequence: GCAGAACGTGGGGCTCACCTC"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     intron          662..1670
                     /label="chimeric intron"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
                     /note="note: chimera between introns from chicken beta-actin and rabbit beta-globin"
     primer_bind     1594..1616
                     /label="pCAGGS-5"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /note="note: Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label="pCAG-F"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /note="note: Rabbit beta-globin intron, for pCAG plasmids, forward primer"
     primer          1729..1777
                     /label="gib-copia-FLAG F"
                     /note="sequence: CAACAGGTTCCCTTTATGCGAAGGGatggactataaggaccacgacgga"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     CDS             1757..1822
                     /label="3xFLAG"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="product: three tandem FLAG(R) epitope tags, followed by anenterokinase cleavage site translation: DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1757..1822
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1823..3742
                     /label="Translation 1823-3742"
                     /translation="TGMFYAHFVLSKRGPLAKIWLAAHWDKKLTKAHVFECNLESSVESIISPKVKMALRTSGHLLLGVVRIYHRKAKYLLADCNEAFIKIKMAFRPGVVDLPEENREAAYNAITLPEEFHDFDQPLPDLDDIDVAQQFSLNQSRVEEITMREEVGNISILQENDFGDFGMDDREIMREGSAFEDDDMLVSTTTSNLLLESEQSTSNLNEKINHLEYEDQYKDDNFGEGNDGGILDDKLISNNDGGIFDDPPALSEAGVMLPEQPAHDDMDEDDNVSMGGPDSPDSVDPVEPMPTMTDQTTLVPNEEEAFALEPIDITVKETKAKRKRKLIVDSVKELDSKTIRAQLSDYSDIVTTLDLAPPTKKLMMWKETGGVEKLFSLPAQPLWNNRLLKLFTRCLTPLVPEDLRKRRKGGEADNLDEFLKEFENPEVPREDQQQQHQQRDVIDEPIIEEPSRLQESVMEASRTNIDESAMPPPPPQGVKRKAGQIDPEPVMPPQQVEQMEIPPVELPPEEPPNICQLIPELELLPEKEKEKEKEKEDDEEEEDEDASGGDQDQEERRWNKRTQQMLHGLQRALAKTGAESISLLELCRNTNRKQAAAKFYSFLVLKKQQAIELTQEEPYSDIIATPGPRFHIILKGGAGG"
     primer          1829..1854
                     /label="gib hRAD21 F"
                     /note="sequence: gattacaaagacgatgacgataagaccggtatgttctacgcacattttgttctcag"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     CDS             1829..3721
                     /label="RAD21 human"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     CDS             1829..3721
                     /label="hRAD21"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="MFYAHFVLSKRGPLAKIWLAAHWDKKLTKAHVFECNLESSVESIISPKVKMALRTSGHLLLGVVRIYHRKAKYLLADCNEAFIKIKMAFRPGVVDLPEENREAAYNAITLPEEFHDFDQPLPDLDDIDVAQQFSLNQSRVEEITMREEVGNISILQENDFGDFGMDDREIMREGSAFEDDDMLVSTTTSNLLLESEQSTSNLNEKINHLEYEDQYKDDNFGEGNDGGILDDKLISNNDGGIFDDPPALSEAGVMLPEQPAHDDMDEDDNVSMGGPDSPDSVDPVEPMPTMTDQTTLVPNEEEAFALEPIDITVKETKAKRKRKLIVDSVKELDSKTIRAQLSDYSDIVTTLDLAPPTKKLMMWKETGGVEKLFSLPAQPLWNNRLLKLFTRCLTPLVPEDLRKRRKGGEADNLDEFLKEFENPEVPREDQQQQHQQRDVIDEPIIEEPSRLQESVMEASRTNIDESAMPPPPPQGVKRKAGQIDPEPVMPPQQVEQMEIPPVELPPEEPPNICQLIPELELLPEKEKEKEKEKEDDEEEEDEDASGGDQDQEERRWNKRTQQMLHGLQRALAKTGAESISLLELCRNTNRKQAAAKFYSFLVLKKQQAIELTQEEPYSDIIATPGPRFHII"
     misc_feature    2573..2575
                     /label="RAD21 misc_feature"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     misc_feature    3188..3190
                     /label="RAD21 misc_feature"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
     misc_feature    3461..3463
                     /label="RAD21 misc_feature"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     primer          3600..3619
                     /label="hRAD21 check F"
                     /note="sequence: aacaagctgccgcaaagttc"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     primer          complement(3696..3721)
                     /label="gib hRAD21 R"
                     /note="sequence: tcacgcctcctgctccccccttaagtataatatggaaccttggtccaggtg"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer          3698..3763
                     /label="gib-link_mCherry F"
                     /note="sequence: atgatcctcagcatgatggaccggcttaaggggggagcaggaggcgtgagcaagggcgaggaggat"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     misc_feature    3722..3742
                     /label="Linker"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
     CDS             3743..4447
                     /label="mCherry"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="product: monomeric derivative of DsRed fluorescent protein(Shaner et al., 2004) note: mammalian codon-optimized translation: VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK"
     CDS             3743..4447
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK"
     primer_bind     complement(3885..3903)
                     /label="mCherry-R"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /note="note: mCherry, reverse primer"
     primer          complement(3885..3903)
                     /label="mCherry int R"
                     /note="sequence: ttggtcaccttcagcttgg"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     primer          complement(4038..4059)
                     /label="mCherry check 5R"
                     /note="sequence: ccgtcctcgaagttcatcacgc"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer_bind     4153..4172
                     /label="mCherry-F"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
                     /note="note: mCherry, forward primer"
     primer          4153..4172
                     /label="mCherry int F"
                     /note="sequence: ccccgtaatgcagaagaaga"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          4370..4393
                     /label="mCherry check 3F"
                     /note="sequence: gaggactacaccatcgtggaacag"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     primer          complement(4426..4481)
                     /label="gib-mCherry NotI R"
                     /note="sequence: ttctgataggcagcctgcacctgagCGGCCGctacttgtacagctcgtccatgccg"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     CDS             4448..4450
                     /label="Translation 4448-4450"
                     /translation="*"
     misc_feature    4457..4481
                     /label="gib HA R"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     primer_bind     complement(4530..4549)
                     /label="Bglob-pA-R"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
                     /note="note: Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4595..4650
                     /label="-beta-globin poly(A) signal"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="note: rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)"
     primer_bind     complement(4649..4668)
                     /label="rbglobpA-R"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
                     /note="note: Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R"
     primer          complement(4736..4760)
                     /label="Inv-Skryre-end:"
                     /note="sequence: ATAGCCACCTTTGTTCATGGCAGCC"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     primer          complement(4736..4760)
                     /label="gib-pA-PGK R"
                     /note="sequence: atagccacctttgttcatggcagcc"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     primer          4783..4805
                     /label="3'-Skryre-out:"
                     /note="sequence: ACAGCCCCCTGCTGTCCATTCCT"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     primer          4921..4945
                     /label="3'-Skryre-mid:"
                     /note="sequence: ACGATGGACTCCAGTCCGGCCCCTCCTCTCCTGACTACTCCCAGTC"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     primer          4999..5024
                     /label="Kostja-VpUC18.F"
                     /note="sequence: gacagaattcgtaatcatggtcatagctgtttcct"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
     primer          complement(5008..5025)
                     /label="Barcode"
                     /note="sequence: catgtaacatgtCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer          complement(5008..5025)
                     /label="Barcode"
                     /note="sequence: taacatgtCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     primer          complement(5008..5025)
                     /label="A10"
                     /note="sequence: CCATCTCATCCCTGCGTGTCTCCGACTCAGTTGAGACTGGGATGTGCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer          complement(5008..5025)
                     /label="Barcode"
                     /note="sequence: tactcgagCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          complement(5008..5025)
                     /label="A11"
                     /note="sequence: CCATCTCATCCCTGCGTGTCTCCGACTCAGGTCGTCTAACGATGTGCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer          complement(5008..5025)
                     /label="A12"
                     /note="sequence: CCATCTCATCCCTGCGTGTCTCCGACTCAGGGTGTCAAGCGATGTGCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer_bind     complement(5009..5025)
                     /label="M13 Reverse"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
                     /note="note: In lacZ gene. Also called M13-rev"
     primer_bind     complement(5009..5025)
                     /label="M13 rev"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="note: common sequencing primer, one of multiple similar variants"
     primer          complement(5009..5025)
                     /label="M13R"
                     /note="sequence: CAGGAAACAGCTATGAC"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     primer_bind     complement(5022..5044)
                     /label="M13/pUC Reverse"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="note: In lacZ gene"
     primer          complement(5022..5045)
                     /label="LIC seq R"
                     /note="sequence: GAGCGGATAACAATTTCACACAGG"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     protein_bind    5033..5049
                     /label="lac operator"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="bound_moiety: lac repressor encoded by lacI note: The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer          complement(5035..5055)
                     /label="Kostja-L-for"
                     /note="sequence: GTGGAATTGTGAGCGGATAAC"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     primer          complement(5037..5056)
                     /label="Kostja-DT-A_seq-f"
                     /note="sequence: tgtggaattgtgagcggata"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     promoter        complement(5057..5087)
                     /label="lac promoter"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="note: promoter for the E. coli lac operon"
     primer          5091..5110
                     /label="Kostja-Int-3F"
                     /note="sequence: tggggtgcctaatgagtgag"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     protein_bind    5102..5123
                     /label="CAP binding site"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="bound_moiety: E. coli catabolite activator protein note: CAP binding activates transcription in the presence of cAMP."
     primer          5183..5205
                     /label="3'-Skryre-in:"
                     /note="sequence: CGACTGCATTAATGAATCGGCCAACG"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     primer_bind     complement(5248..5265)
                     /label="L4440"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="note: L4440 vector, forward primer"
     primer          5311..5333
                     /label="5'-Molder-out"
                     /note="sequence: GCATGGCGGTAATACGGTTATCCACAG"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer          complement(5331..5354)
                     /label="TandemPolyA seq R"
                     /note="sequence: ttcctgcgttatcccctgattctg"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer          complement(5334..5363)
                     /label="Kostja-gBlock3-r"
                     /note="sequence: ACATGTTCTTNCCTGCGTTATCCCCTGATT"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer          5340..5365
                     /label="Kostja-pUC-Ori-f"
                     /note="sequence: GGGATAACGCAGGAAAGAACATGTGA"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     primer          5353..5382
                     /label="Kostja-Ori-f"
                     /note="sequence: AAAGAACATGNGAGCAAAAGGCCAGCAAAA"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     rep_origin      complement(5419..6007)
                     /label="ori"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="direction: LEFT note: high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
     primer          5438..5457
                     /label="integOri_L"
                     /note="sequence: CCTGACGAGCATCACAAAAA"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer          5458..5480
                     /label="5'-Molder-mid"
                     /note="sequence: TCGACGCTCAAGTCAGAGGTGGC"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     primer          complement(5466..5485)
                     /label="Kostja-Ori-3R"
                     /note="sequence: gtttcgccacctctgacttg"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer_bind     complement(5499..5518)
                     /label="pBR322ori-F"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
                     /note="note: pBR322 origin, forward primer"
     primer          5521..5542
                     /label="5'-Molder-inn"
                     /note="sequence: TGGAAGCTCCCTCGTGCGCTCT"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     primer          complement(5728..5747)
                     /label="integOri_R"
                     /note="sequence: GTCTTACCGGGTTGGACTCA"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          complement(5794..5817)
                     /label="3'-Molder-inn"
                     /note="sequence: GTAGCACCGCCTACATACCTCGCT"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          complement(5798..5817)
                     /label="Kostja-Ori-2R"
                     /note="sequence: gtagcaccgcctacatacct"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer          complement(5869..5892)
                     /label="3'-Molder-mid"
                     /note="sequence: GGCTTCAGCAGAGCGCAGATACCA"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer          complement(5969..5992)
                     /label="3'-Molder-out"
                     /note="sequence: TCTGCGCGTAATCTGCTGCTTGCA"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          6018..6041
                     /label="123_loc_1"
                     /note="sequence: tgatcttttctacggggtctgacg"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     primer          complement(6063..6078)
                     /label=111111
                     /note="sequence: ttAataatctcatAaccaaaatcccttaac"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     primer          6159..6183
                     /label="pvui-amp-end"
                     /note="sequence: agtaaacttggtctgacagttacca"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     CDS             complement(6178..7038)
                     /label="AmpR"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="gene: bla product: beta-lactamase note: confers resistance to ampicillin, carbenicillin, andrelated antibiotics translation: MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     CDS             complement(6178..7038)
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     primer          6194..6214
                     /label="Amp_3_endR"
                     /note="sequence: agtgaggcacctatctcagcg"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          6395..6415
                     /label="qpTandemAmpRF"
                     /note="sequence: GCAACTTTATCCGCCTCCATC"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     primer          complement(6503..6522)
                     /label="qpTandemAmpRR"
                     /note="sequence: AACGACGAGCGTGACACCAC"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     primer_bind     6801..6820
                     /label="Amp-R"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="note: Ampicillin resistance gene, reverse primer"
     primer          complement(7001..7021)
                     /label="TAIL308-0(full)"
                     /note="sequence: ccgtgtcgcccttattccctt"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     promoter        complement(7039..7143)
                     /label="AmpR promoter"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="gene: bla"
     primer          7071..7100
                     /label="Kostja-gBlock4-f"
                     /note="sequence: ATCAGGGTTANTGTCTCATGAGCGGATACA"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcctcgag aattctcacg
     1741 cgtgaattcc accatggact ataaggacca cgacggagac tacaaggatc atgatattga
     1801 ttacaaagac gatgacgata agaccggtat gttctacgca cattttgttc tcagtaaaag
     1861 agggcctctg gccaaaattt ggctagcggc ccattgggat aagaagctaa ccaaagccca
     1921 tgtgttcgag tgtaatttag agagcagcgt ggagagtatc atctcaccaa aggtgaaaat
     1981 ggcattacgg acatcaggac atctcttact gggagtagtt cgaatctatc acaggaaagc
     2041 caaatacctt cttgcagact gtaatgaagc attcattaag ataaagatgg cttttcggcc
     2101 aggtgtggtt gacctgcctg aggaaaatcg ggaagcagct tataatgcca ttactttacc
     2161 tgaagaattt catgactttg atcagccact gcctgactta gatgacatcg atgtggccca
     2221 gcagttcagc ttgaatcaga gtagagtgga agagataacc atgagagaag aagttgggaa
     2281 catcagtatt ttacaagaaa atgattttgg tgattttgga atggatgatc gtgagataat
     2341 gagagaaggc agtgcttttg aggatgacga catgttagta agcactacta cttctaacct
     2401 cctattagag tctgaacaga gcaccagcaa tctgaatgag aaaattaacc atttagaata
     2461 tgaagatcaa tataaggatg ataattttgg agaaggaaat gatggtggaa tattagatga
     2521 caaacttatt agtaataatg atggcggtat ctttgatgat ccccctgccc tctctgaggc
     2581 aggggtgatg ttgccagagc agcctgcaca tgacgatatg gatgaggatg ataatgtatc
     2641 aatgggtggg cctgatagtc ctgattcagt ggatcccgtt gaaccaatgc caaccatgac
     2701 tgatcaaaca acacttgttc caaatgagga agaagcattt gcattggagc ctattgatat
     2761 aactgttaaa gaaacaaaag ccaagaggaa gaggaagcta attgttgaca gtgtcaaaga
     2821 gttggatagc aagacaatta gagcccaact tagtgattat tcagatattg ttactacttt
     2881 ggatctggca ccgcccacca agaaattgat gatgtggaaa gagacaggag gagtagaaaa
     2941 actgttttct ttacctgctc agcctttgtg gaataacaga ctactgaagc tctttacacg
     3001 ctgtcttaca ccgcttgtac cagaagacct tagaaaaagg aggaaaggag gagaggcaga
     3061 taatttggat gaattcctca aagaatttga aaatccagag gttcctagag aggaccagca
     3121 acagcagcat cagcagcgtg atgttatcga tgagcccatt attgaagagc caagccgcct
     3181 ccaggagtca gtgatggagg ccagcagaac aaacatagat gagtcagcta tgcctccacc
     3241 accacctcag ggagttaagc gaaaagctgg acaaattgac ccagagcctg tgatgcctcc
     3301 tcagcaggta gagcagatgg aaataccacc tgtagagctt cccccagaag aacctccaaa
     3361 tatctgtcag ctaataccag agttagaact tctgccagaa aaagagaagg agaaagagaa
     3421 ggaaaaagaa gatgatgaag aggaagagga tgaagatgca tcagggggcg atcaagatca
     3481 ggaagaaaga agatggaaca aaaggactca gcagatgctt catggtcttc agcgtgctct
     3541 tgctaaaact ggagctgaat ctatcagttt gcttgagtta tgtcgaaata cgaacagaaa
     3601 acaagctgcc gcaaagttct acagcttctt ggttcttaaa aagcagcaag ctattgagct
     3661 gacacaggaa gaaccgtaca gtgacatcat cgcaacacct ggaccaaggt tccatattat
     3721 acttaagggg ggagcaggag gcgtgagcaa gggcgaggag gataacatgg ccatcatcaa
     3781 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
     3841 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     3901 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc
     3961 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
     4021 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     4081 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
     4141 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg
     4201 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga
     4261 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct
     4321 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac
     4381 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct
     4441 gtacaagtag CGGCCGctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
     4501 aatgccctgg ctcacaaata ccactgagat ctttttccct ctgccaaaaa ttatggggac
     4561 atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca
     4621 atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat
     4681 ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccat atgctggctg
     4741 ccatgaacaa aggtggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca
     4801 ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt
     4861 gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt
     4921 cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgaa gatccctcga
     4981 cctgcagccc aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5041 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5101 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5161 acctgtcgtg ccagcggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5221 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5281 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5341 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5401 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5461 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5521 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5581 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
     5641 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5701 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5761 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5821 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     5881 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5941 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6001 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6061 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6121 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6181 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6241 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6301 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6361 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6421 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6481 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6541 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6601 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6661 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6721 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6781 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6841 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6901 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6961 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7021 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7081 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7141 gcgcacattt ccccgaaaag tgccacctg
//