LOCUS       sequence-350685-        6286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Viral vector for co-expression of non-functional NaChBac and EGFP in
            cells expressing Cre AND NOT Flp driven by the human Synapsin 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6286)
  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 6286)
  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-mNaChBacMUT-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..6286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    78..98
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        120..567
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    567..591
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(598..631)
                     /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(685..718)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    725..758
                     /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(802..1515,1516..1518,1519..1521))
                     /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(805..1515,1516..1518,1519..1521))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(847..868)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1196..1215
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1455..1476
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1522..1578)
                     /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    2467..2500
                     /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    2510..2543
                     /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    2594..2627
                     /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(2628..2652)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2662..3250
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2715..2735)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3133..3144)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3280..3297)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3286..3493
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3501..3641
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3501..3630
                     /label=AAV2 ITR
     rep_origin      3716..4171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3803..3822)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4013..4034
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4188..4207)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4307..4329
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4367..4385)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4453..4557
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4558..4626,4627..5418)
                     /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(4776..4795)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5589..6177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6078..6097
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcccgggcg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
       61 taggggttcc ttctagacaa ctttgtatag aaaagttgct gcagagggcc ctgcgtatga
      121 gtgcaagtgg gttttaggac caggatgagg cggggtgggg gtgcctacct gacgaccgac
      181 cccgacccac tggacaagca cccaaccccc attccccaaa ttgcgcatcc cctatcagag
      241 agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg
      301 acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg
      361 ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc
      421 gccgccgccg gcccagccgg accgcaccac gcgaggcgcg agataggggg gcacgggcgc
      481 gaccatctgc gctgcggcgc cggcgactca gcgctgcctc agtctgcggt gggcagcgga
      541 ggagtcgtgt cgtgcctgag agcgcagcaa gtttgtacaa aaaagcaggc tgccaccata
      601 acttcgtata ggatacttta tacgaagtta tccggcagaa tggtagctgg attgtagctg
      661 ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg
      721 cgcggaagtt cctattctct agaaagtata ggaacttccc tcgagtaccg gtcacgcgta
      781 cgcaagcgcc ttagcggccg cttacttgta cagctcgtcc atgccgagag tgatcccggc
      841 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
      901 ggcggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
      961 ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat
     1021 cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga cgttgtggct
     1081 gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
     1141 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
     1201 gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc
     1261 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc
     1321 gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
     1381 gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa
     1441 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
     1501 ctcctcgccc ttgctcacca taggcccggg gttttcttca acatctcctg cttgctttaa
     1561 cagagagaag ttcgtggctc cgcttcctcc actgccgata tccttaagtt gtttgagcaa
     1621 gctcttcagt tcggctacat cctttcgcaa tgctgaaatc tcctgtttaa gtccgtcggc
     1681 ctccccatct tcctcattgt ccgtaagctc cgccttttcc acgttgttca caatgactcc
     1741 gatgaaaaga ttgaaaatga taaaagtacc aatcaggaca aacgagacaa agtagagcca
     1801 tgaccagggc acctctgcaa aaatgggccg cattacaccg ctggcccagc ttttcaaggt
     1861 caccacttgg aacagcgtca gcaacgaaag ctggaggttc ccaaagtatt caggcgatac
     1921 gtgctggaag agcattgtcc cgatcactgc aaagatatag aagaagatgg acatcaagat
     1981 caagatattg cccaacgccg gaatcgtcat caccagcgca tctacgagtc tgcgcaggga
     2041 ggggacgacg gagatcgccc tgagtacccg cagcactcta aggattcgca atactgtcac
     2101 gaactgggct cccgcgaaga tatgcccagc cgcgacaatc agaaaatcaa accaattcca
     2161 cgagctgcgg aagaaggccg actttgggtt cgacgccaaa aacctcatcg cgatctcgat
     2221 ggtgaagatc caaagcagga cgaggtcaat ccgataaaag agccacttgt ggtccgcgta
     2281 gatcctcgga tatgtttcga ttcccacgat cagtgcatta aagaggatca gggcaatgac
     2341 ggtgaaagta aacgctctat ggttgacaat tttctgcatc tttgacgtga atgagttctg
     2401 tttctggcgt gcttccattt tcatggtggc ggatcctagg tacctgagct caacgatagc
     2461 actagtgaag ttcctattct ctagaaagta taggaacttc gctagccgga taacttcgta
     2521 taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt tattctttag
     2581 aatggtgcaa agaataactt cgtataatgt atgctatacg aagttatacc cagctttctt
     2641 gtacaaagtg ggaattccga taatcaacct ctggattaca aaatttgtga aagattgact
     2701 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     2761 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     2821 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     2881 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     2941 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3001 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     3061 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     3121 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     3181 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     3241 gcctccccgc atcgggaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt
     3301 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3361 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3421 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca
     3481 ggcatgctgg ggagggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     3541 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     3601 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     3661 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     3721 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3781 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3841 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3901 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     3961 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4021 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     4081 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4141 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     4201 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4261 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4321 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     4381 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     4441 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4501 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4561 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4621 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4681 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4741 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4801 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4861 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4921 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4981 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5041 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5101 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5161 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5221 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc
     5281 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5341 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5401 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5461 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5521 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5581 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5641 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5701 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5761 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5821 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5881 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5941 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6001 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6061 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6121 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6181 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc
     6241 tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcaa
//
