LOCUS       pCAG-3XFLAG-brRAD21-mCH 7238 bp ds-DNA     circular     14-JAN-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"
     primer          1829..1853
                     /label="gib brRAD21 F"
                     /note="sequence: gattacaaagacgatgacgataagaccggtatgttYtaYgcccatttcatcYtga"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     CDS             1829..3790
                     /label="RAD21 Branchiostoma lanceolatum"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     CDS             1829..3790
                     /label="BraLan RAD21"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="MFYAHFILSKKGPLAKVWLAAHWDKKLTKQHVFETSIEGSVESILHPKVKMALRTSGHLLLGVVRIHNRKAKYLLADCNEAFVKIKMAFRPGVVDLPEENREAAVATITLPEVFHDFDTTVPDLNDFDMQAFTMNQSRVEEITMREDVGNINMMETDAFGDLPFDDKEMLRDASNMDETLYNTADASNLLLEGDHNKKDDTVEKRHPELRYDDPIKDDFGGAPGDGILGADFLGATAGDLFGEAPGGPTGDISIGPEIAATPIPEQANDKERAGPSGELQPDQNATAVADQTTLVPNEREAFALEPLEVTGVKETRVRKKRKLIVDPVIELASSEIRAQLEDTSDIVTTLDLAPPTKKLMQWKEMGGVDKLFTMPCRAVGAKVLLAIFHRNLNTVIPEDLREEKGSAVDDEERMEIESTEMGRDDLYGESFLSYFFKIGGSSINILDDGPMSFGGPASVSSLGSVAEPLPMGDDDDDDLPPKSVEPPEVPERRSSPTPEEQAVVSKNSKASPSKRKNALGGVEDLFGQATGSPAVDLPPMSPVKTPSSSPRKAYRTPRGGGDSSHANETEEEQEERRLNKRTTQMLHVMDRTMGSTGSITFFDMTKKHTRKQAAAKFYTLLVLKKQTAIEVEQKEHFSDILITKGPKFNSLVGA"
     primer          3622..3643
                     /label="brRAD21 check F"
                     /note="sequence: catcacgttcttcgacatgacg"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     primer          3713..3734
                     /label="brRAD21 check F2"
                     /note="sequence: atcgaagtcgaacagaaggaac"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer          3767..3832
                     /label="gib-link_mCherry F"
                     /note="sequence: atgatcctcagcatgatggaccggcttaaggggggagcaggaggcgtgagcaagggcgaggaggat"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     primer          complement(3769..3790)
                     /label="gib brRAD21 R"
                     /note="sequence: tcacgcctcctgctccccccttaagcgcYccgactaaactgttaaac"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     misc_feature    3791..3791
                     /label="gib HA L"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     misc_feature    3791..3811
                     /label="Linker"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
     CDS             3812..4516
                     /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             3812..4516
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK"
     primer_bind     complement(3954..3972)
                     /label="mCherry-R"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /note="note: mCherry, reverse primer"
     primer          complement(3954..3972)
                     /label="mCherry int R"
                     /note="sequence: ttggtcaccttcagcttgg"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     primer          complement(4107..4128)
                     /label="mCherry check 5R"
                     /note="sequence: ccgtcctcgaagttcatcacgc"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer_bind     4222..4241
                     /label="mCherry-F"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
                     /note="note: mCherry, forward primer"
     primer          4222..4241
                     /label="mCherry int F"
                     /note="sequence: ccccgtaatgcagaagaaga"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          4439..4462
                     /label="mCherry check 3F"
                     /note="sequence: gaggactacaccatcgtggaacag"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     primer          complement(4495..4550)
                     /label="gib-mCherry NotI R"
                     /note="sequence: ttctgataggcagcctgcacctgagCGGCCGctacttgtacagctcgtccatgccg"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     CDS             4517..4519
                     /label="Translation 4517-4519"
                     /translation="*"
     misc_feature    4526..4550
                     /label="gib HA R"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     primer_bind     complement(4599..4618)
                     /label="Bglob-pA-R"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
                     /note="note: Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4664..4719
                     /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(4718..4737)
                     /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(4805..4829)
                     /label="Inv-Skryre-end:"
                     /note="sequence: ATAGCCACCTTTGTTCATGGCAGCC"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     primer          complement(4805..4829)
                     /label="gib-pA-PGK R"
                     /note="sequence: atagccacctttgttcatggcagcc"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     primer          4852..4874
                     /label="3'-Skryre-out:"
                     /note="sequence: ACAGCCCCCTGCTGTCCATTCCT"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     primer          4990..5014
                     /label="3'-Skryre-mid:"
                     /note="sequence: ACGATGGACTCCAGTCCGGCCCCTCCTCTCCTGACTACTCCCAGTC"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     primer          5068..5093
                     /label="Kostja-VpUC18.F"
                     /note="sequence: gacagaattcgtaatcatggtcatagctgtttcct"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
     primer          complement(5077..5094)
                     /label="Barcode"
                     /note="sequence: taacatgtCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     primer          complement(5077..5094)
                     /label="Barcode"
                     /note="sequence: tactcgagCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          complement(5077..5094)
                     /label="A11"
                     /note="sequence: CCATCTCATCCCTGCGTGTCTCCGACTCAGGTCGTCTAACGATGTGCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer          complement(5077..5094)
                     /label="A10"
                     /note="sequence: CCATCTCATCCCTGCGTGTCTCCGACTCAGTTGAGACTGGGATGTGCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer          complement(5077..5094)
                     /label="A12"
                     /note="sequence: CCATCTCATCCCTGCGTGTCTCCGACTCAGGGTGTCAAGCGATGTGCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer          complement(5077..5094)
                     /label="Barcode"
                     /note="sequence: catgtaacatgtCAGGAAACAGCTATGACC"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer_bind     complement(5078..5094)
                     /label="M13 Reverse"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
                     /note="note: In lacZ gene. Also called M13-rev"
     primer_bind     complement(5078..5094)
                     /label="M13 rev"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="note: common sequencing primer, one of multiple similar variants"
     primer          complement(5078..5094)
                     /label="M13R"
                     /note="sequence: CAGGAAACAGCTATGAC"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     primer_bind     complement(5091..5113)
                     /label="M13/pUC Reverse"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="note: In lacZ gene"
     primer          complement(5091..5114)
                     /label="LIC seq R"
                     /note="sequence: GAGCGGATAACAATTTCACACAGG"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     protein_bind    5102..5118
                     /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(5104..5124)
                     /label="Kostja-L-for"
                     /note="sequence: GTGGAATTGTGAGCGGATAAC"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     primer          complement(5106..5125)
                     /label="Kostja-DT-A_seq-f"
                     /note="sequence: tgtggaattgtgagcggata"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     promoter        complement(5126..5156)
                     /label="lac promoter"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="note: promoter for the E. coli lac operon"
     primer          5160..5179
                     /label="Kostja-Int-3F"
                     /note="sequence: tggggtgcctaatgagtgag"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     protein_bind    5171..5192
                     /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          5252..5274
                     /label="3'-Skryre-in:"
                     /note="sequence: CGACTGCATTAATGAATCGGCCAACG"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     primer_bind     complement(5317..5334)
                     /label="L4440"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="note: L4440 vector, forward primer"
     primer          5380..5402
                     /label="5'-Molder-out"
                     /note="sequence: GCATGGCGGTAATACGGTTATCCACAG"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer          complement(5400..5423)
                     /label="TandemPolyA seq R"
                     /note="sequence: ttcctgcgttatcccctgattctg"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer          complement(5403..5432)
                     /label="Kostja-gBlock3-r"
                     /note="sequence: ACATGTTCTTNCCTGCGTTATCCCCTGATT"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer          5409..5434
                     /label="Kostja-pUC-Ori-f"
                     /note="sequence: GGGATAACGCAGGAAAGAACATGTGA"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     primer          5422..5451
                     /label="Kostja-Ori-f"
                     /note="sequence: AAAGAACATGNGAGCAAAAGGCCAGCAAAA"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     rep_origin      complement(5488..6076)
                     /label="ori"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="direction: LEFT note: high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
     primer          5507..5526
                     /label="integOri_L"
                     /note="sequence: CCTGACGAGCATCACAAAAA"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer          5527..5549
                     /label="5'-Molder-mid"
                     /note="sequence: TCGACGCTCAAGTCAGAGGTGGC"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     primer          complement(5535..5554)
                     /label="Kostja-Ori-3R"
                     /note="sequence: gtttcgccacctctgacttg"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     primer_bind     complement(5568..5587)
                     /label="pBR322ori-F"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
                     /note="note: pBR322 origin, forward primer"
     primer          5590..5611
                     /label="5'-Molder-inn"
                     /note="sequence: TGGAAGCTCCCTCGTGCGCTCT"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     primer          complement(5797..5816)
                     /label="integOri_R"
                     /note="sequence: GTCTTACCGGGTTGGACTCA"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          complement(5863..5886)
                     /label="3'-Molder-inn"
                     /note="sequence: GTAGCACCGCCTACATACCTCGCT"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          complement(5867..5886)
                     /label="Kostja-Ori-2R"
                     /note="sequence: gtagcaccgcctacatacct"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer          complement(5938..5961)
                     /label="3'-Molder-mid"
                     /note="sequence: GGCTTCAGCAGAGCGCAGATACCA"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     primer          complement(6038..6061)
                     /label="3'-Molder-out"
                     /note="sequence: TCTGCGCGTAATCTGCTGCTTGCA"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          6087..6110
                     /label="123_loc_1"
                     /note="sequence: tgatcttttctacggggtctgacg"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     primer          complement(6132..6147)
                     /label=111111
                     /note="sequence: ttAataatctcatAaccaaaatcccttaac"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     primer          6228..6252
                     /label="pvui-amp-end"
                     /note="sequence: agtaaacttggtctgacagttacca"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     CDS             complement(6247..7107)
                     /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(6247..7107)
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     primer          6263..6283
                     /label="Amp_3_endR"
                     /note="sequence: agtgaggcacctatctcagcg"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer          6464..6484
                     /label="qpTandemAmpRF"
                     /note="sequence: GCAACTTTATCCGCCTCCATC"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     primer          complement(6572..6591)
                     /label="qpTandemAmpRR"
                     /note="sequence: AACGACGAGCGTGACACCAC"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     primer_bind     6870..6889
                     /label="Amp-R"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="note: Ampicillin resistance gene, reverse primer"
     primer          complement(7070..7090)
                     /label="TAIL308-0(full)"
                     /note="sequence: ccgtgtcgcccttattccctt"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     promoter        complement(7108..7212)
                     /label="AmpR promoter"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="gene: bla"
     primer          7140..7169
                     /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 gttctacgcc catttcatct tgagtaagaa
     1861 gggccccctg gcaaaggtgt ggctggctgc tcattgggac aagaagctta ccaagcagca
     1921 tgtgtttgag actagcatcg aaggcagtgt ggagagcatc cttcatccaa aggtgaagat
     1981 ggccttacga acatcaggcc atctgttgct gggagtggtg cgtatccaca accgcaaggc
     2041 caagtaccta ctggctgact gcaacgaagc cttcgtcaag atcaagatgg cgttccggcc
     2101 tggcgtggtg gatctcccgg aggagaaccg tgaagcggct gtggcgacca tcactcttcc
     2161 cgaggtgttc cacgacttcg acaccaccgt tcctgacttg aatgattttg acatgcaggc
     2221 tttcaccatg aaccagagcc gtgtggaaga aatcaccatg agagaggacg tcggcaacat
     2281 caacatgatg gagacggacg ccttcggtga tcttcctttt gacgataagg aaatgttgcg
     2341 agatgccagc aacatggacg agactttgta caacacggcg gacgccagca acctcctgct
     2401 ggagggcgat cacaacaaga aggacgacac ggtagagaag cgccacccgg agctcagata
     2461 cgatgacccc atcaaggacg actttggcgg ggcccccgga gacggtatcc taggtgctga
     2521 tttcctgggt gctacagcag gggacttgtt cggagaggct ccaggggggc cgaccggcga
     2581 catctccatc gggcctgaga tagctgcaac gcccatccct gaacaggcta atgacaagga
     2641 acgcgcaggt cctagcggtg agctacaacc ggaccagaac gcaaccgctg tcgccgacca
     2701 gacaactctg gtaccaaacg aacgggaggc ttttgcttta gagcctctag aagtcacagg
     2761 tgtcaaagaa accagggtgc gtaagaagcg gaagctgatc gtggacccgg tgatcgagct
     2821 ggccagctcg gagatccgtg cccagctgga ggacacctcg gacatcgtca cgacccttga
     2881 cctggccccg cccaccaaga agctgatgca atggaaggag atgggcggcg tggacaagct
     2941 gttcaccatg ccgtgtcgag ctgttggggc aaaagtcttg ctagccattt tccacagaaa
     3001 tttgaacacc gtaatcccag aagacctgag ggaagagaag ggatcggcag tggacgatga
     3061 agaacgaatg gagatcgagt caacagaaat gggaagagat gacttgtatg gtgagtcctt
     3121 tttgtcatat ttctttaaga taggtggttc ctcgatcaac atccttgacg acggtccgat
     3181 gtcgttcggc ggtccagcgt cagtgtccag tctgggaagt gttgcggagc cgctaccaat
     3241 gggagacgac gatgatgacg accttcctcc caagtccgta gaaccaccgg aggtgcccga
     3301 gagaaggtcg tcacctacgc cagaagaaca ggctgttgtt agtaaaaaca gtaaagcttc
     3361 gccgtcaaag cgcaagaacg cgctgggtgg agtagaagac ctgtttggac aggcaactgg
     3421 tagccctgct gtggacctcc ccccaatgtc accagtcaag acaccatcat catcacccag
     3481 aaaggcgtac cgtactccaa gaggcggtgg tgactcctct cacgcgaacg agacagagga
     3541 agagcaggag gagagacgac tgaacaaacg taccacacag atgcttcacg tcatggacag
     3601 aaccatgggc agcaccggtt ccatcacgtt cttcgacatg acgaaaaagc acacccggaa
     3661 acaggccgcc gccaaattct acaccctact ggtcctgaag aaacagaccg cgatcgaagt
     3721 cgaacagaag gaacatttca gtgatatcct cattacaaag ggaccaaagt ttaacagttt
     3781 agtcggggcg cttaaggggg gagcaggagg cgtgagcaag ggcgaggagg ataacatggc
     3841 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     3901 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     3961 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     4021 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     4081 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     4141 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     4201 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     4261 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     4321 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     4381 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     4441 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     4501 ggacgagctg tacaagtagC GGCCGctcag gtgcaggctg cctatcagaa ggtggtggct
     4561 ggtgtggcca atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat
     4621 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     4681 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg
     4741 gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgccata
     4801 tgctggctgc catgaacaaa ggtggctata aagaggtcat cagtatatga aacagccccc
     4861 tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt
     4921 ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc
     4981 cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc tcttatgaag
     5041 atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5101 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5161 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5221 agtcgggaaa cctgtcgtgc cagcggatcc gctgcattaa tgaatcggcc aacgcgcggg
     5281 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5341 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5401 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5461 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5521 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5581 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5641 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta
     5701 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5761 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5821 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5881 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     5941 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6001 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6061 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6121 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6181 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6241 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6301 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6361 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6421 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6481 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6541 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6601 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6661 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6721 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6781 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6841 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6901 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6961 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7021 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7081 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7141 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7201 aggggttccg cgcacatttc cccgaaaagt gccacctg
//