LOCUS       sequence-310155-        7505 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 7505)
  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 7505)
  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..7505
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1765..1830
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(4063..4065,4066..4776)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4063..4065,4066..4776)
                     /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(4105..4126)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4366..4385)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4713..4734
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4864..4883)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4929..4984
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4983..5002)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5345..5361)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5345..5361)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5358..5380)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5369..5385
                     /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(5393..5399,5400..5417,5418..5423))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5438..5459
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5584..5601)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5755..6343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5835..5854)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6514..7305,7306..7374))
                     /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     7137..7156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7375..7479)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggsgcgc gccrggsggg gsggggsggg gsgrggggsg
      541 gggsggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcctcgagaa
     1741 ttctcacgcg tgaattccac catggactat aaggaccacg acggagacta caaggatcat
     1801 gatattgatt acaaagacga tgacgataag accggtgaag gtgaggcggt tgaagccatt
     1861 gtggaggagt ctgaaacttt cattaaagga aaagaaagaa agacttacca gagacgccgg
     1921 gaagggggcc aggaagaaga tgcttgccac ctgccccaga accagacaga tgggggtgag
     1981 gtggtccagg atgtcaacag cagtgtacag atggtaatga tggaacagct ggatcctacc
     2041 cttctccaga tgaagactga agtcatggag ggtacagtgg ctccggaagc agaggctgca
     2101 gtggacgata cccagatcat aaccttgcag gttgtaaata tggaggaaca gcccattaac
     2161 ataggagagc ttcagcttgt ccaagtacct gttcctgtga cggtacctgt tgctactact
     2221 tcagtagaag aacttcaggg ggcttatgag aatgaagtgt ctaaagaggg ccttgcagaa
     2281 agtgaaccga tgatatgtca caccttacct ttgcctgaag gatttcaggt ggtgaaagtg
     2341 ggggccaatg gagaagtgga gacactagag cagggcgagc ttcctcctca ggaagactct
     2401 agctggcaaa aagacccaga ctatcagcca ccagccaaaa aaacaaagaa aaccaaaaag
     2461 agcaaacttc gttacacaga agagggcaaa gacgtggatg tgtctgtgta tgattttgag
     2521 gaagaacagc aggaaggact gctgtctgag gttaatgcag agaaagtagt tggtaatatg
     2581 aagcctccga agccaacaaa aattaaaaaa aaaggtgtaa agaaaacatt ccagtgtgag
     2641 ctttgcagtt acacatgtcc ccggcgttca aatttggatc gtcacatgaa aagccacact
     2701 gatgagagac cacacaaatg ccatctgtgt ggcagagcat tcagaacagt gaccctcctg
     2761 aggaatcatc tgaacacaca cacaggtact cgtcctcaca agtgcccaga ctgcgatatg
     2821 gcctttgtga ccagtggaga attggtgcgg catcgtcgtt ataaacacac tcatgagaaa
     2881 ccatttaagt gttccatgtg tgattatgcc agtgtagaag tcagcaaatt aaaacgacac
     2941 attcgctctc atactggaga gcgcccgttc cagtgcagtt tgtgcagtta tgccagcagg
     3001 gacacataca agctgaaaag gcatatgaga acccattcag gggaaaaacc ttatgaatgt
     3061 tatatttgtc acgctcggtt tacccagagt ggtaccatga agatgcacat tttacagaag
     3121 cacacagaaa atgtggccaa atttcattgt ccccattgtg acactgtcat agcccgaaaa
     3181 agtgatttgg gtgtccactt gcgaaagcag cattcctata ttgaacaggg caaaaaatgt
     3241 cgctactgtg atgctgtgtt tcatgagcga tatgctctca tccagcatca aaaatcacac
     3301 aaaaatgaga agcgcttcaa gtgtgaccag tgtgattatg cttgtagaca ggagcggcac
     3361 atgatcatgc acaagcgcac tcacacgggg gagaagcctt atgcctgcag ccactgcgac
     3421 aagaccttcc gccagaaaca gctcctcgac atgcatttca agcgctatca tgatcccaac
     3481 tttgtccctg ctgcctttgt ctgttccaag tgtgggaaaa cattcacccg ccggaacaca
     3541 atggcaagac atgcagataa ctgtgctggt ccagatggcg tagaggggga aaatggaggg
     3601 gagacaaaga agagcaaacg aggaagaaaa agaaagatgc gatctaaaaa agaagactcc
     3661 tctgacagtg aagaaaatgc tgagccggat ctcgatgaca atgaggagga ggaggagcct
     3721 gctgtagaaa ttgaacctga gccagagcct cagccccagc ctccgcctcc acctcagcct
     3781 gtggccccgg cccccccacc tgccaagaag agaagggggc gacctcctgg aagaaccaac
     3841 cagcccaaac agaaccagcc aacagccatc attcaggtcg aagatcagaa tacaggtgca
     3901 attgagaaca ttatagttga agtcaaaaag gagccagatg ccgagcctgc ggagggggaa
     3961 gaagaggagg ctcaggcagc caccacagac gcccccaacg gagacctcac gcctgagatg
     4021 atcctcagca tgatggaccg gcttaagggg ggagcaggag gcgtgagcaa gggcgaggag
     4081 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     4141 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     4201 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     4261 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     4321 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     4381 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     4441 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     4501 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     4561 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     4621 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     4681 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4741 gccgggatca ctctcggcat ggacgagctg tacaagtcgc gataagctag ctcaggtgca
     4801 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     4861 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     4921 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     4981 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     5041 ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag
     5101 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
     5161 gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct
     5221 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
     5281 catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc ttggcgtaat
     5341 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5401 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5461 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgct
     5521 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5581 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5641 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5701 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5761 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5821 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5881 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5941 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6001 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6061 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6121 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6181 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6241 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6301 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6361 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6421 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6481 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6541 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6601 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6661 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6721 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6781 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6841 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6901 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6961 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7021 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7081 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7141 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7201 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7261 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7321 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7381 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7441 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7501 acctg
//
