LOCUS       sequence-441121-        5840 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 5840)
  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 5840)
  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-CAG-FLEX-ArgiNLS-EGFP
FEATURES             Location/Qualifiers
     source          1..5840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    347..468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(384..403)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     438..457
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    511..1099
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             617..628
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1026..1046
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    1141..1174
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1245..1278
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             join(1376..1378,1379..2092)
                     /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(1376..1378,1379..2089)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1418..1439)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1679..1698)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2026..2047
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2119..2152)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(2215..2248)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(2283..2302)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(2364..2386)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(2665..2942)
                     /label=chicken beta-actin promoter
     repeat_region   3253..3382
                     /label=AAV2 ITR
     rep_origin      3454..3909
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3541..3560)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3751..3772
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3935..4039
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4040..4108,4109..4900)
                     /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(4258..4277)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5071..5659
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5560..5579
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg
       61 cgctcgctcg ctcactgagg ccgcccgggc aarrscmrsk cgkcsgscgm mctttggtcg
      121 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg
      181 ttccttgtag ttaatgatta acccgccatg ctacttatct acgtagccat gctctaggaa
      241 gatccttatc gattttacca catttgtaga ggttttactt gctttaaaaa acctcccaca
      301 tctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
      361 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
      421 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgctc
      481 gaagcggccg cccgggtcga cggtatcgat gcggggaggc ggcccaaagg gagatccgac
      541 tcgtctgagg gcgaaggcga agacgcggaa gaggccgcag agccggcagc aggccgcggg
      601 aaggaaggtc cgctggattg agggccgaag ggacgtagca gaaggacgtc ccgcgcagaa
      661 tccaggtggc aacacaggcg agcagccaag gaaaggacga tgatttcccc gacaacacca
      721 cggaattgtc agtgcccaac agccgagccc ctgtccagca gcgggcaagg caggcggcga
      781 tgagttccgc cgtggcaata gggaggggga aagcgaaagt cccggaaagg agctgacagg
      841 tggtggcaat gccccaacca gtgggggttg cgtcagcaaa cacagtgcac accacgccac
      901 gttgcctgac aacgggccac aactcctcat aaagagacag caaccaggat ttatacaagg
      961 aggagaaaat gaaagccata cgggaagcaa tagcatgata caaaggcatt aaagcagcgt
     1021 atccacatag cgtaaaagga gcaacatagt taagaatacc agtcaatctt tcacaaattt
     1081 tgtaatccag aggttgatta tcgataagct tgatatcgaa ttcgaacctc ttcgagggac
     1141 ataacttcgt ataatgtatg ctatacgaag ttatgggtcg atggtgagat ctggactaga
     1201 gggtcgatgg tgatgcttgg atcaaaatag gaagaatccc aatgataact tcgtataaag
     1261 tatcctatac gaagttatac tagaattcgg taccgccacc atgaaaaggg tggctcccca
     1321 gaagcagatg agcatagcaa agaaaaagaa ggtgagaagg cggcggcgcc gtcgagtgag
     1381 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1441 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     1501 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     1561 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     1621 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     1681 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1741 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1801 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1861 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1921 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1981 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     2041 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aagatatcct
     2101 cgagggggga tccctagtat aacttcgtat agcatacatt atacgaagtt atgggtcgat
     2161 ggtgatgctt ggcaattcgg gtcgatggtg aagcattggt cttcctattt tgatataact
     2221 tcgtatagga tactttatac gaagttatgg ggatccaatt ctttgccaaa atgatgagac
     2281 agcacaataa ccagcacgtt gcccaggagc tgtaggaaaa agaagaaggc atgaacatgg
     2341 ttagcagagg ctctagagcc gccggtcaca cgccagaagc cgaaccccgc cctgccccgt
     2401 cccccccgaa ggcagccgtc cccccgcgga cagccccgag ccgctccccc cgcacaaagg
     2461 gccctcccgg agcccctcaa ggctttcacg cagccacaga aaagaaacaa gccgtcatta
     2521 aaccaagcgc taattacagc ccggaggaga agggccgtcc cgcccgctca cctgtgggag
     2581 taacgcggtc agtcagagcc ggggcgggcg gcgcgaggcg gcggcggagc ggggcacggg
     2641 gcgaaggcag cgcgcagcga ctcccgcccg ccgcgcgctt cgctttttat agggccgccg
     2701 ccgccgccgc ctcgccataa aaggaaactt tcggagcgcg ccgctctgat tggctgccgc
     2761 cgcacctctc cgcctcgccc cgccccgccc ctcgccccgc cccgccccgc ctggcgcgcg
     2821 cccccccccc ccccccgccc ccatcgctgc acaaaataat taaaaaataa ataaatacaa
     2881 aattgggggt ggggaggggg gggagatggg gagagtgaag cagaacgtgg ggctcacctc
     2941 gaccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa
     3001 ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg
     3061 cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc
     3121 cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat
     3181 tgacgtcaat ggtactcttc ctagagcatg gctacgtaga taagtagcat ggcgggttaa
     3241 tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     3301 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     3361 cagtgagcga gcgagcgcgc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     3421 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     3481 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3541 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3601 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3661 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3721 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3781 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3841 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     3901 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3961 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4021 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4081 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4141 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4201 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4261 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4321 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4381 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     4441 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     4501 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     4561 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     4621 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     4681 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     4741 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     4801 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4861 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4921 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4981 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5041 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5101 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5161 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5221 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5281 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5341 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5401 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5461 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5521 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5581 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5641 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5701 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     5761 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     5821 agtgagcgag gaagcggaag
//
