LOCUS       sequence-301983-        5308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For transient expression of nuclear eGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5308)
  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 5308)
  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: pEN716 - pCAGGS-eGFP-3Xnls-bpA
FEATURES             Location/Qualifiers
     source          1..5308
                     /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             join(1754..1756,1757..1759,1760..2470)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1754..1756,1757..1759,1760..2470)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1799..1820)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2060..2079)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2407..2428
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2504..2524
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2528..2548
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2552..2572
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2669..2688)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2734..2789
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2788..2807)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3148..3164)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3148..3164)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3161..3183)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3172..3188
                     /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(3196..3202,3203..3220,3221..3226))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3241..3262
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3387..3404)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3558..4146)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3638..3657)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4317..5108,5109..5177))
                     /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     4940..4959
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5178..5282)
                     /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 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     1801 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     1861 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1921 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     1981 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     2041 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     2101 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2161 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     2221 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     2281 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2341 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     2401 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2461 gctgtacaag tcgcgaatgg ccacaaccat ggtgcacgtg gatccaaaaa agaagagaaa
     2521 ggtagatcca aaaaagaaga gaaaggtaga tccaaaaaag aagagaaagg tacacgtgtc
     2581 ccccgggtaa gcggcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca
     2641 atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca
     2701 tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa
     2761 tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt
     2821 taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgccata tgctggctgc
     2881 catgaacaaa ggtggctata aagaggtcat cagtatatga aacagccccc tgctgtccat
     2941 tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg
     3001 ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc
     3061 ctcctctcct gactactccc agtcatagct gtccctcttc tcttatgaag atccctcgac
     3121 ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3181 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3241 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3301 cctgtcgtgc cagcggatcc gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3361 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3421 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3481 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3541 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3601 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3661 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3721 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
     3781 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3841 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3901 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3961 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     4021 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4081 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4141 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4201 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4261 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4321 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4381 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4441 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4501 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4561 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4621 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4681 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4741 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4801 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4861 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4921 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4981 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5041 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5101 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5161 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5221 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5281 cgcacatttc cccgaaaagt gccacctg
//
