LOCUS       sequence-310163-        7481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For transient expression of human CTCF tagged with eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7481)
  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 7481)
  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: pKS070 - pCAGGS-3XFLAG-(human)CTCF-eGFP
FEATURES             Location/Qualifiers
     source          1..7481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     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(4039..4041,4042..4752)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4039..4041,4042..4752)
                     /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(4081..4102)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4342..4361)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4689..4710
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4840..4859)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4905..4960
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4959..4978)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5321..5337)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5321..5337)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5334..5356)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5345..5361
                     /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(5369..5375,5376..5393,5394..5399))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5414..5435
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5560..5577)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5731..6319)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5811..5830)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6490..7281,7282..7350))
                     /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     7113..7132
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7351..7455)
                     /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 gggsggggsg 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 accggtatgg aaggtgatgc agtcgaagcc
     1861 attgtggagg agtccgaaac ttttattaaa ggaaaggaga gaaagactta ccagagacgc
     1921 cgggaagggg gccaggaaga agatgcctgc cacttacccc agaaccagac ggatgggggt
     1981 gaggtggtcc aggatgtcaa cagcagtgta cagatggtga tgatggaaca gctggacccc
     2041 acccttcttc agatgaagac tgaagtaatg gagggcacag tggctccaga agcagaggct
     2101 gctgtggacg atacccagat tataacttta caggttgtaa atatggagga acagcccata
     2161 aacataggag aacttcagct tgttcaagta cctgttcctg tgactgtacc tgttgctacc
     2221 acttcagtag aagaacttca gggggcttat gaaaatgaag tgtctaaaga gggccttgcg
     2281 gaaagtgaac ccatgatatg ccacacccta cctttgcctg aagggtttca ggtggttaaa
     2341 gtgggggcca atggagaggt ggagacacta gaacaagggg aacttccacc ccaggaagat
     2401 cctagttggc aaaaagaccc agactatcag ccaccagcca aaaaaacaaa gaaaaccaaa
     2461 aagagcaaac tgcgttatac agaggagggc aaagatgtag atgtgtctgt ctacgatttt
     2521 gaggaagaac agcaggaggg tctgctatca gaggttaatg cagagaaagt ggttggtaat
     2581 atgaagcctc caaagccaac aaaaattaaa aagaaaggtg taaagaagac attccagtgt
     2641 gagctttgca gttacacgtg tccacggcgt tcaaatttgg atcgtcacat gaaaagccac
     2701 actgatgaga gaccacacaa gtgccatctc tgtggcaggg cattcagaac agtcaccctc
     2761 ctgaggaatc accttaacac acacacaggt actcgtcctc acaagtgccc agactgcgac
     2821 atggcctttg tgaccagtgg agaattggtt cggcatcgtc gttacaaaca cacccacgag
     2881 aagccattca agtgttccat gtgcgattac gccagtgtag aagtcagcaa attaaaacgt
     2941 cacattcgct ctcatactgg agagcgtccg tttcagtgca gtttgtgcag ttatgccagc
     3001 agggacacat acaagctgaa aaggcacatg agaacccatt caggggaaaa gccttatgaa
     3061 tgttatattt gtcatgctcg gtttacccaa agtggtacca tgaagatgca cattttacag
     3121 aagcacacag aaaatgtggc caaatttcac tgtccccact gtgacacagt catagcccga
     3181 aaaagtgatt tgggtgtcca cttgcgaaag cagcattcct atattgagca aggcaagaaa
     3241 tgccgttact gtgatgctgt gtttcatgag cgctatgccc tcatccagca tcagaagtca
     3301 cacaagaatg agaagcgctt taagtgtgac cagtgtgatt acgcttgtag acaggagagg
     3361 cacatgatca tgcacaagcg cacccacacc ggggagaagc cttacgcctg cagccactgc
     3421 gataagacct tccgccagaa gcagcttctc gacatgcact tcaagcgcta tcacgacccc
     3481 aacttcgtcc ctgcggcttt tgtctgttct aagtgtggga aaacatttac acgtcggaat
     3541 accatggcaa gacatgctga taattgtgct ggcccagatg gcgtagaggg ggaaaatgga
     3601 ggagaaacga agaagagtaa acgtggaaga aaaagaaaga tgcgctctaa gaaagaagat
     3661 tcctctgaca gtgaaaatgc tgaaccagat ctggacgaca atgaggatga ggaggagcct
     3721 gccgtagaaa ttgaacctga gccagagcct cagcctgtga ccccagcccc accacccgcc
     3781 aagaagcgga gaggacgacc ccctggcaga accaaccagc ccaaacagaa ccagccaaca
     3841 gctatcattc aggttgaaga ccagaataca ggtgcaattg agaacattat agttgaagta
     3901 aaaaaagagc cagatgctga gcccgcagag ggagaggaag aggaggccca gccagctgcc
     3961 acagatgccc ccaacggaga cctcacgccc gagatgatcc tcagcatgat ggaccggctt
     4021 aaggggggag caggaggcgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     4081 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     4141 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     4201 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     4261 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     4321 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     4381 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     4441 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     4501 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     4561 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4621 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     4681 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     4741 gagctgtaca agtcgcgata agctagctca ggtgcaggct gcctatcaga aggtggtggc
     4801 tggtgtggcc aatgccctgg ctcacaaata ccactgagat ctttttccct ctgccaaaaa
     4861 ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat
     4921 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag
     4981 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca
     5041 tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc
     5101 ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat
     5161 attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact
     5221 agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg
     5281 gagatccctc gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     5341 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     5401 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     5461 tccagtcggg aaacctgtcg tgccagcgga tccgctgcat taatgaatcg gccaacgcgc
     5521 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5581 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5641 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5701 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5761 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5821 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5881 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5941 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6001 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6061 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6121 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6181 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6241 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6301 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6361 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6421 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6481 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6541 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6601 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6661 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6721 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6781 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6841 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6901 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6961 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7021 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7081 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7141 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7201 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7261 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7321 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7381 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7441 aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
