LOCUS       sequence-301985-        7471 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 7471)
  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 7471)
  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..7471
                     /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(4031..4033,4034..4744)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4031..4033,4034..4744)
                     /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(4073..4094)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4334..4353)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4681..4702
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4832..4851)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4897..4952
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4951..4970)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5311..5327)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5311..5327)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5324..5346)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5335..5351
                     /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(5359..5365,5366..5383,5384..5389))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5404..5425
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5550..5567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5721..6309)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5801..5820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6480..7271,7272..7340))
                     /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     7103..7122
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7341..7445)
                     /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 agaccggtat ggaaggtgat gcagtcgaag ccattgtgga
     1861 ggagtccgaa acttttatta aaggaaagga gagaaagact taccagagac gccgggaagg
     1921 gggccaggaa gaagatgcct gccacttacc ccagaaccag acggatgggg gtgaggtggt
     1981 ccaggatgtc aacagcagtg tacagatggt gatgatggaa cagctggacc ccacccttct
     2041 tcagatgaag actgaagtaa tggagggcac agtggctcca gaagcagagg ctgctgtgga
     2101 cgatacccag attataactt tacaggttgt aaatatggag gaacagccca taaacatagg
     2161 agaacttcag cttgttcaag tacctgttcc tgtgactgta cctgttgcta ccacttcagt
     2221 agaagaactt cagggggctt atgaaaatga agtgtctaaa gagggccttg cggaaagtga
     2281 acccatgata tgccacaccc tacctttgcc tgaagggttt caggtggtta aagtgggggc
     2341 caatggagag gtggagacac tagaacaagg ggaacttcca ccccaggaag atcctagttg
     2401 gcaaaaagac ccagactatc agccaccagc caaaaaaaca aagaaaacca aaaagagcaa
     2461 actgcgttat acagaggagg gcaaagatgt agatgtgtct gtctacgatt ttgaggaaga
     2521 acagcaggag ggtctgctat cagaggttaa tgcagagaaa gtggttggta atatgaagcc
     2581 tccaaagcca acaaaaatta aaaagaaagg tgtaaagaag acattccagt gtgagctttg
     2641 cagttacacg tgtccacggc gttcaaattt ggatcgtcac atgaaaagcc acactgatga
     2701 gagaccacac aagtgccatc tctgtggcag ggcattcaga acagtcaccc tcctgaggaa
     2761 tcaccttaac acacacacag gtactcgtcc tcacaagtgc ccagactgcg acatggcctt
     2821 tgtgaccagt ggagaattgg ttcggcatcg tcgttacaaa cacacccacg agaagccatt
     2881 caagtgttcc atgtgcgatt acgccagtgt agaagtcagc aaattaaaac gtcacattcg
     2941 ctctcatact ggagagcgtc cgtttcagtg cagtttgtgc agttatgcca gcagggacac
     3001 atacaagctg aaaaggcaca tgagaaccca ttcaggggaa aagccttatg aatgttatat
     3061 ttgtcatgct cggtttaccc aaagtggtac catgaagatg cacattttac agaagcacac
     3121 agaaaatgtg gccaaatttc actgtcccca ctgtgacaca gtcatagccc gaaaaagtga
     3181 tttgggtgtc cacttgcgaa agcagcattc ctatattgag caaggcaaga aatgccgtta
     3241 ctgtgatgct gtgtttcatg agcgctatgc cctcatccag catcagaagt cacacaagaa
     3301 tgagaagcgc tttaagtgtg accagtgtga ttacgcttgt agacaggaga ggcacatgat
     3361 catgcacaag cgcacccaca ccggggagaa gccttacgcc tgcagccact gcgataagac
     3421 cttccgccag aagcagcttc tcgacatgca cttcaagcgc tatcacgacc ccaacttcgt
     3481 ccctgcggct tttgtctgtt ctaagtgtgg gaaaacattt acacgtcgga ataccatggc
     3541 aagacatgct gataattgtg ctggcccaga tggcgtagag ggggaaaatg gaggagaaac
     3601 gaagaagagt aaacgtggaa gaaaaagaaa gatgcgctct aagaaagaag attcctctga
     3661 cagtgaaaat gctgaaccag atctggacga caatgaggat gaggaggagc ctgccgtaga
     3721 aattgaacct gagccagagc ctcagcctgt gaccccagcc ccaccacccg ccaagaagcg
     3781 gagaggacga ccccctggca gaaccaacca gcccaaacag aaccagccaa cagctatcat
     3841 tcaggttgaa gaccagaata caggtgcaat tgagaacatt atagttgaag taaaaaaaga
     3901 gccagatgct gagcccgcag agggagagga agaggaggcc cagccagctg ccacagatgc
     3961 ccccaacgga gacctcacgc ccgagatgat cctcagcatg atggaccggc ttaagggggg
     4021 agcaggaggc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     4081 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     4141 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     4201 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     4261 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     4321 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     4381 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     4441 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     4501 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     4561 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     4621 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     4681 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     4741 caagtcgcga taagctagct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg
     4801 ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg
     4861 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     4921 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     4981 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc atatgctggc
     5041 tgccatgaac aaaggtggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc
     5101 cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt
     5161 gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact agccagattt
     5221 ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg aagatccctc
     5281 gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5341 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5401 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5461 aaacctgtcg tgccagcgga tccgctgcat taatgaatcg gccaacgcgc ggggagaggc
     5521 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5581 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5641 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5701 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5761 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5821 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5881 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
     5941 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6001 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6061 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6121 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     6181 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6241 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6301 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6361 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6421 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6481 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6541 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6601 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6661 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6721 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6781 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6841 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6901 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6961 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7021 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7081 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7141 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7201 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7261 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7321 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7381 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7441 ccgcgcacat ttccccgaaa agtgccacct g
//
