LOCUS       sequence-350687-        5562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Viral vector for co-expression of a CDS of interest and EGFP in 
            cells expressing Cre AND NOT Flp driven by a Synapsin promoter. 
            Contains an in-frame P2A sequence and an MCS for cloning of the 
            CDS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5562)
  AUTHORS   Ubina T, Vahedi-Hunter T, Agnew-Svoboda W, Wong W, Gupta A, 
            Santhakumar V, Riccomagno MM
  TITLE     ExBoX - a simple Boolean exclusion strategy to drive expression in 
            neurons.
  JOURNAL   J Cell Sci. 2021 Oct 15;134(20). pii: 272538. doi: 
            10.1242/jcs.257212. Epub 2021 Oct 20.
  PUBMED    34515305
REFERENCE   2  (bases 1 to 5562)
  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: "J Cell 
            Sci."; date: "2021-10-15"; volume: "134(20). pii: 272538. doi"; 
            pages: " 10.1242/jcs.257212. Epub 2021 Oct 20"
COMMENT     Sequence Label: pAAV-SynI-CreOn-FlpOff-MCS-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..5562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(55..72)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    61..268
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   276..416
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   276..405
                     /label=AAV2 ITR
     rep_origin      491..946
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(578..597)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     788..809
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(963..982)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1082..1104
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1142..1160)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1228..1332
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1333..1401,1402..2193)
                     /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(1551..1570)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2364..2952
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2853..2872
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   3014..3154
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3025..3154
                     /label=AAV2 ITR
     protein_bind    3161..3181
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        3203..3650
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    3650..3674
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3681..3714)
                     /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)."
     protein_bind    complement(3768..3801)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3808..3841
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(join(3885..4598,4599..4601,4602..4604))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(3888..4598,4599..4601,4602..4604))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3930..3951)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4279..4298
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4538..4559
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(4605..4661)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    4804..4837
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    4847..4880
                     /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)."
     protein_bind    4931..4964
                     /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(4965..4989)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(4999..5562,1..25)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5052..5072)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5470..5481)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 atctcccttt gggccgcctc cccgcatcgg gaattcctag agctcgctga tcagcctcga
       61 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
      121 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
      181 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
      241 gggaagagaa tagcaggcat gctggggagg gccgcaggaa cccctagtga tggagttggc
      301 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
      361 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
      421 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
      481 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
      541 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
      601 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
      661 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
      721 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
      781 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
      841 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
      901 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
      961 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     1021 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     1081 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     1141 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     1201 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1261 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     1321 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     1381 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     1441 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     1501 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     1561 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     1621 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     1681 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     1741 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     1801 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     1861 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     1921 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     1981 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     2041 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     2101 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     2161 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     2221 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     2281 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2341 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2401 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2461 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     2521 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2581 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2641 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2701 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2761 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2821 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2881 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2941 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3001 tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggca
     3061 aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga
     3121 gagggagtgg ccaactccat cactaggggt tccttctaga caactttgta tagaaaagtt
     3181 gctgcagagg gccctgcgta tgagtgcaag tgggttttag gaccaggatg aggcggggtg
     3241 ggggtgccta cctgacgacc gaccccgacc cactggacaa gcacccaacc cccattcccc
     3301 aaattgcgca tcccctatca gagaggggga ggggaaacag gatgcggcga ggcgcgtgcg
     3361 cactgccagc ttcagcaccg cggacagtgc cttcgccccc gcctggcggc gcgcgccacc
     3421 gccgcctcag cactgaaggc gcgctgacgt cactcgccgg tcccccgcaa actccccttc
     3481 ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc cggaccgcac cacgcgaggc
     3541 gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg cgccggcgac tcagcgctgc
     3601 ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct gagagcgcag caagtttgta
     3661 caaaaaagca ggctgccacc ataacttcgt ataggatact ttatacgaag ttatccggca
     3721 gaatggtagc tggattgtag ctgctattag caatatgaaa cctcttaata acttcgtata
     3781 gcatacatta tacgaagtta tggcgcggaa gttcctattc tctagaaagt ataggaactt
     3841 ccctcgagta ccggtcacgc gtacgcaagc gccttagcgg ccgcttactt gtacagctcg
     3901 tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc
     3961 ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt gtcgggcagc
     4021 agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg
     4081 ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg
     4141 gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg
     4201 ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg
     4261 aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc
     4321 tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg
     4381 tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg
     4441 ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc
     4501 tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg
     4561 atgggcacca ccccggtgaa cagctcctcg cccttgctca ccataggccc ggggttttct
     4621 tcaacatctc ctgcttgctt taacagagag aagttcgtgg ctccgcttcc tctagagggg
     4681 taccgtcgac ccaagcttcc cgggagaaca ggttacccac cactgcagat ctcccactgt
     4741 taattaagtg aagcaggccg gccgcgggga tcctaggtac ctgagctcaa cgatagcact
     4801 agtgaagttc ctattctcta gaaagtatag gaacttcgct agccggataa cttcgtataa
     4861 agtatcctat acgaagttat ttgccttaac ccagaaatta tcactgttat tctttagaat
     4921 ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag ttatacccag ctttcttgta
     4981 caaagtggga attccgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     5041 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     5101 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     5161 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     5221 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     5281 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     5341 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg
     5401 tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     5461 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     5521 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc gg
//
