LOCUS       sequence-441212-        5451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of a single-cell discriminating version of 
            EGFP fluorescent protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5451)
  AUTHORS   Szelenyi ER, Navarrete JS, Murry AD, Zhang Y, Girven KS, Kuo L, 
            Cline MM, Bernstein MX, Burdyniuk M, Bowler B, Goodwin NL, Juarez B,
            Zweifel LS, Golden SA
  TITLE     An arginine-rich nuclear localization signal (ArgiNLS) strategy for 
            streamlined image segmentation of single cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Aug 6;121(32):e2320250121. doi: 
            10.1073/pnas.2320250121. Epub 2024 Jul 29.
  PUBMED    39074275
REFERENCE   2  (bases 1 to 5451)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-08-6"; volume: "121(32):e2320250121. 
            doi"; pages: " 10.1073/pnas.2320250121. Epub 2024 Jul 29"
COMMENT     Sequence Label: pAAV-EF1a-ArgiNLS-EGFP
FEATURES             Location/Qualifiers
     source          1..5451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(1521..1523,1524..2237)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1521..1523,1524..2234)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1563..1584)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1824..1843)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2171..2192
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2313..2333)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2715..2855
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2715..2844
                     /label=AAV2 ITR
     rep_origin      2930..3385
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3017..3036)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3227..3248
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3402..3421)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3521..3543
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3581..3599)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3667..3771
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3772..3840,3841..4632)
                     /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     complement(3990..4009)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4803..5390
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5291..5310
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccgtacc ggatcctcta gagggtaccg
     1441 ccaccatgaa aagggtggct ccccagaagc agatgagcat agcaaagaaa aagaaggtga
     1501 gaaggcggcg gcgccgtcga gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1561 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1621 agggcgatgc cacctacggc aagctgaccc tgaagttcat ttgcaccacc ggcaagctgc
     1681 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1741 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1801 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1861 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1921 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1981 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     2041 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2101 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     2161 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2221 acgagctgta caagtaggat atcgaattcc gctcgagata atcaacctct ggattacaaa
     2281 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     2341 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     2401 ttgtataaat cctggttagt tcttgccacg gcggaactca tcgccgcctg ccttgcccgc
     2461 tgctggacag gggctcggct gttgggcact gacaattccg tggtgtttat ttgtgaaatt
     2521 tgtgatgcta ttgctttatt tgtaaccatc tagctttatt tgtgaaattt gtgatgctat
     2581 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     2641 ttttatgttt caggttcagg gggagatgtg ggaggttttt taaagcggcc gccacgtgcg
     2701 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     2761 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     2821 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     2881 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     2941 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3001 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3061 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3121 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     3181 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3241 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     3301 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3361 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     3421 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3481 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3541 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     3601 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     3661 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3721 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3781 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3841 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3901 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3961 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4021 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4081 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4141 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4201 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4261 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4321 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4381 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4441 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4501 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4561 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4621 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4681 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4741 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4801 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4861 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4921 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4981 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5041 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5101 aaggcgcagc ggtcgggctg aacgggggtt cgtgcacaca gcccagcttg gagcgaacga
     5161 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5221 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5281 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5341 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5401 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
