LOCUS       sequence-310130-        5318 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 5318)
  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 5318)
  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..5318
                     /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             join(1762..1764,1765..1767,1768..2478)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1762..1764,1765..1767,1768..2478)
                     /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(1807..1828)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2068..2087)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2415..2436
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2512..2532
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2536..2556
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2560..2580
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2677..2696)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2742..2797
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2796..2815)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3158..3174)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3158..3174)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3171..3193)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3182..3198
                     /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(3206..3212,3213..3230,3231..3236))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3251..3272
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3397..3414)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3568..4156)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3648..3667)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4327..5118,5119..5187))
                     /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     4950..4969
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5188..5292)
                     /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 rgggctcggg 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 catggtgagc aagggcgagg agctgttcac cggggtggtg
     1801 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     1861 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     1921 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     1981 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     2041 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     2101 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2161 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     2221 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     2281 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     2341 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     2401 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     2461 atggacgagc tgtacaagtc gcgaatggcc acaaccatgg tgcacgtgga tccaaaaaag
     2521 aagagaaagg tagatccaaa aaagaagaga aaggtagatc caaaaaagaa gagaaaggta
     2581 cacgtgtccc ccgggtaagc ggcctcaggt gcaggctgcc tatcagaagg tggtggctgg
     2641 tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta
     2701 tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt
     2761 cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc
     2821 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat
     2881 gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc
     2941 tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt
     3001 ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc
     3061 cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag
     3121 atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3181 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3241 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3301 agtcgggaaa cctgtcgtgc cagcggatcc gctgcattaa tgaatcggcc aacgcgcggg
     3361 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3421 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3481 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3541 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3601 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3661 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3721 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3781 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3841 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3901 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3961 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4021 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4081 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4141 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4201 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4261 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4321 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4381 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4441 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4501 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4561 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4621 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4681 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4741 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4801 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4861 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4921 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4981 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5041 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5101 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5161 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5221 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5281 aggggttccg cgcacatttc cccgaaaagt gccacctg
//
