LOCUS       sequence-245710-        7802 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive, ubiquitous, nuclear-localized GFP expression. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7802)
  AUTHORS   Tang W, Das A, Pegoraro AF, Han YL, Huang J, Roberts DA, Yang H, 
            Fredberg JJ, Kotton DN, Bi D, Guo M
  TITLE     Collective curvature sensing and fluidity in three-dimensional 
            multicellular systems
  JOURNAL   Nat. Phys. 18, 1371–1378 (2022).
REFERENCE   2  (bases 1 to 7802)
  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. Phys. 
            18, 1371–1378 (2022)."
COMMENT     Sequence Label: pHAGE-EF1aL-nlsGFP-W
FEATURES             Location/Qualifiers
     source          1..7802
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2223..3401
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2454..3392
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3349..3369
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3406..3415
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3418..3438
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3439..3441,3442..4152)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3439..3441,3442..4152)
                     /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(3481..3502)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3742..3761)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4089..4110
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4153..4170
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    4192..4780
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4245..4265)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4663..4674)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4855..5088
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5207..5225)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5293..5397
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5398..5466,5467..6258)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5616..5635)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6429..7017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6918..6937
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7171..7188
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7428..7449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7464..7469,7470..7487,7488..7494)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7502..7518
                     /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)."
     primer_bind     7507..7529
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7526..7542
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7526..7542
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7592..7611
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtcgt
     2221 gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     2281 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
     2341 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     2401 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg
     2461 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa
     2521 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
     2581 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
     2641 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
     2701 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
     2761 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
     2821 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
     2881 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
     2941 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
     3001 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
     3061 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
     3121 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
     3181 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
     3241 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
     3301 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
     3361 cagtggttca aagttttttt cttccatttc aggtgtcgtg aagcggccgc catggccccc
     3421 aagaagaagc gcaaggtggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     3481 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     3541 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     3601 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     3661 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     3721 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     3781 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     3841 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     3901 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     3961 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4021 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     4081 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     4141 gagctgtaca agcatcatca ccatcaccat tgactcgagg gcccagatcc taatcaacct
     4201 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     4261 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     4321 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     4381 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     4441 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     4501 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     4561 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     4621 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     4681 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     4741 cctcagacga gtcggatctc cctttgggcc gcctccccgc ctgagatcct ttaagaccaa
     4801 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     4861 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
     4921 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     4981 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     5041 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat
     5101 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagaggc
     5161 ccgggttaat taaggaaagg gctagatcat tcttgaagac gaaagggcct cgtgatacgc
     5221 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5281 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5341 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5401 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5461 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5521 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5581 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5641 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5701 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5761 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5821 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5881 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5941 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6001 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6061 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6121 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6181 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6241 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6301 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6361 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6421 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6481 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6541 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6601 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6661 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6721 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6781 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6841 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6901 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6961 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7021 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7081 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7141 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     7201 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagcaagctc
     7261 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     7321 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctccccgtgg
     7381 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     7441 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     7501 attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac gaatttcaca
     7561 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     7621 tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc
     7681 ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac ctgtgattcc
     7741 tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac aaacttagta
     7801 gt
//
