LOCUS       sequence-301987-        7495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For transient expression of mouse CTCF tagged with eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7495)
  AUTHORS   Nora EP, Caccianini L, Fudenberg G, So K, Kameswaran V, Nagle A, 
            Uebersohn A, Hajj B, Saux AL, Coulon A, Mirny LA, Pollard KS, Dahan 
            M, Bruneau BG
  TITLE     Molecular basis of CTCF binding polarity in genome folding.
  JOURNAL   Nat Commun. 2020 Nov 5;11(1):5612. doi: 10.1038/s41467-020-19283-x.
  PUBMED    33154377
REFERENCE   2  (bases 1 to 7495)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2020-11-5"; volume: "11(1):5612. doi"; pages: " 
            10.1038/s41467-020-19283-x"
COMMENT     Sequence Label: pKS004 - pCAGGS-3XFLAG-CTCF-eGFP
FEATURES             Location/Qualifiers
     source          1..7495
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1757..1822
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(4055..4057,4058..4768)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4055..4057,4058..4768)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4097..4118)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4358..4377)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4705..4726
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4856..4875)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4921..4976
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4975..4994)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5335..5351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5335..5351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5348..5370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5359..5375
                     /label=lac operator
                     /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)."
     promoter        complement(join(5383..5389,5390..5407,5408..5413))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5428..5449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5574..5591)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5745..6333)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5825..5844)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6504..7295,7296..7364))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7127..7146
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7365..7469)
                     /gene="bla"
                     /label=AmpR promoter
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 agaccggtga aggtgaggcg gttgaagcca ttgtggagga
     1861 gtctgaaact ttcattaaag gaaaagaaag aaagacttac cagagacgcc gggaaggggg
     1921 ccaggaagaa gatgcttgcc acctgcccca gaaccagaca gatgggggtg aggtggtcca
     1981 ggatgtcaac agcagtgtac agatggtaat gatggaacag ctggatccta cccttctcca
     2041 gatgaagact gaagtcatgg agggtacagt ggctccggaa gcagaggctg cagtggacga
     2101 tacccagatc ataaccttgc aggttgtaaa tatggaggaa cagcccatta acataggaga
     2161 gcttcagctt gtccaagtac ctgttcctgt gacggtacct gttgctacta cttcagtaga
     2221 agaacttcag ggggcttatg agaatgaagt gtctaaagag ggccttgcag aaagtgaacc
     2281 gatgatatgt cacaccttac ctttgcctga aggatttcag gtggtgaaag tgggggccaa
     2341 tggagaagtg gagacactag agcagggcga gcttcctcct caggaagact ctagctggca
     2401 aaaagaccca gactatcagc caccagccaa aaaaacaaag aaaaccaaaa agagcaaact
     2461 tcgttacaca gaagagggca aagacgtgga tgtgtctgtg tatgattttg aggaagaaca
     2521 gcaggaagga ctgctgtctg aggttaatgc agagaaagta gttggtaata tgaagcctcc
     2581 gaagccaaca aaaattaaaa aaaaaggtgt aaagaaaaca ttccagtgtg agctttgcag
     2641 ttacacatgt ccccggcgtt caaatttgga tcgtcacatg aaaagccaca ctgatgagag
     2701 accacacaaa tgccatctgt gtggcagagc attcagaaca gtgaccctcc tgaggaatca
     2761 tctgaacaca cacacaggta ctcgtcctca caagtgccca gactgcgata tggcctttgt
     2821 gaccagtgga gaattggtgc ggcatcgtcg ttataaacac actcatgaga aaccatttaa
     2881 gtgttccatg tgtgattatg ccagtgtaga agtcagcaaa ttaaaacgac acattcgctc
     2941 tcatactgga gagcgcccgt tccagtgcag tttgtgcagt tatgccagca gggacacata
     3001 caagctgaaa aggcatatga gaacccattc aggggaaaaa ccttatgaat gttatatttg
     3061 tcacgctcgg tttacccaga gtggtaccat gaagatgcac attttacaga agcacacaga
     3121 aaatgtggcc aaatttcatt gtccccattg tgacactgtc atagcccgaa aaagtgattt
     3181 gggtgtccac ttgcgaaagc agcattccta tattgaacag ggcaaaaaat gtcgctactg
     3241 tgatgctgtg tttcatgagc gatatgctct catccagcat caaaaatcac acaaaaatga
     3301 gaagcgcttc aagtgtgacc agtgtgatta tgcttgtaga caggagcggc acatgatcat
     3361 gcacaagcgc actcacacgg gggagaagcc ttatgcctgc agccactgcg acaagacctt
     3421 ccgccagaaa cagctcctcg acatgcattt caagcgctat catgatccca actttgtccc
     3481 tgctgccttt gtctgttcca agtgtgggaa aacattcacc cgccggaaca caatggcaag
     3541 acatgcagat aactgtgctg gtccagatgg cgtagagggg gaaaatggag gggagacaaa
     3601 gaagagcaaa cgaggaagaa aaagaaagat gcgatctaaa aaagaagact cctctgacag
     3661 tgaagaaaat gctgagccgg atctcgatga caatgaggag gaggaggagc ctgctgtaga
     3721 aattgaacct gagccagagc ctcagcccca gcctccgcct ccacctcagc ctgtggcccc
     3781 ggccccccca cctgccaaga agagaagggg gcgacctcct ggaagaacca accagcccaa
     3841 acagaaccag ccaacagcca tcattcaggt cgaagatcag aatacaggtg caattgagaa
     3901 cattatagtt gaagtcaaaa aggagccaga tgccgagcct gcggaggggg aagaagagga
     3961 ggctcaggca gccaccacag acgcccccaa cggagacctc acgcctgaga tgatcctcag
     4021 catgatggac cggcttaagg ggggagcagg aggcgtgagc aagggcgagg agctgttcac
     4081 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     4141 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     4201 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     4261 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     4321 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     4381 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     4441 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     4501 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     4561 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     4621 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     4681 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     4741 cactctcggc atggacgagc tgtacaagtc gcgataagct agctcaggtg caggctgcct
     4801 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     4861 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     4921 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
     4981 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
     5041 tggcaacata tgccatatgc tggctgccat gaacaaaggt ggctataaag aggtcatcag
     5101 tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt
     5161 tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc
     5221 ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc
     5281 cctcttctct tatgaagatc cctcgacctg cagcccaagc ttggcgtaat catggtcata
     5341 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5401 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5461 ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgct gcattaatga
     5521 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5581 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5641 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5701 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5761 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5821 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5881 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     5941 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6001 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6061 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6121 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6181 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6241 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6301 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6361 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6421 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6481 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6541 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6601 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6661 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6721 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6781 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6841 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6901 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6961 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7021 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7081 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7141 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7201 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7261 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7321 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7381 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7441 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctg
//
