LOCUS       sequence-344910-        7455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-on / Flp-off helpers for N2c rabies monosynaptic tracing; TVA, 
            eGFP and N2cG under Synpasin promotoer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7455)
  AUTHORS   Pouchelon G, Dwivedi D, Bollmann Y, Agba CK, Xu Q, Mirow AMC, Kim S,
            Qiu Y, Sevier E, Ritola KD, Cossart R, Fishell G
  TITLE     The organization and development of cortical interneuron presynaptic
            circuits are area specific.
  JOURNAL   Cell Rep. 2021 Nov 9;37(6):109993. doi: 
            10.1016/j.celrep.2021.109993.
  PUBMED    34758329
REFERENCE   2  (bases 1 to 7455)
  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: "Cell Rep.";
            date: "2021-11-9"; volume: "37(6):109993. doi"; pages: " 
            10.1016/j.celrep.2021.109993"
COMMENT     Sequence Label: pAAV-VTKS5-TVA-eGFP-N2cG
FEATURES             Location/Qualifiers
     source          1..7455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   8..148
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8..137
                     /label=AAV2 ITR
     rep_origin      223..678
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(310..329)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     520..541
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(695..714)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     814..836
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(874..892)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        960..1064
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1065..1133,1134..1925)
                     /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(1283..1302)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2096..2684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2585..2604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2746..2875
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2903..3350
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    3374..3407
                     /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    complement(3414..3447)
                     /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(3498..3531)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(5127..5183)
                     /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"
     CDS             complement(join(5193..5903,5904..5906,5907..5909))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(5193..5903,5904..5906,5907..5909))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5235..5256)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5584..5603
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5843..5864
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(5910..5966)
                     /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"
     regulatory      6443..6452
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6481..6514
                     /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    6565..6598
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    6599..6632
                     /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)"
     misc_feature    6641..7229
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6694..6714)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7112..7123)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    join(7237..7455,1..6)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 ctatggcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
       61 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
      121 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
      181 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
      241 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
      301 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      361 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      421 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
      481 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
      541 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
      601 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
      661 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
      721 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
      781 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
      841 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
      901 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
      961 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1021 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     1081 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     1141 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     1201 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     1261 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     1321 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     1381 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     1441 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     1501 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     1561 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     1621 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     1681 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     1741 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     1801 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     1861 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     1921 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     1981 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     2041 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2101 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2161 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2221 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     2281 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2341 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2401 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2461 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2521 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2581 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2641 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2701 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtcctgc aggcagctgc
     2761 gcgctcgctc gctcactgag gccgcccggg cgtcgggcga cctttggtcg cccggcctca
     2821 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgctag
     2881 actgcagagg gccctgcgta tgagtgcaag tgggttttag gaccaggatg aggcggggtg
     2941 ggggtgccta cctgacgacc gaccccgacc cactggacaa gcacccaacc cccattcccc
     3001 aaattgcgca tcccctatca gagaggggga ggggaaacag gatgcggcga ggcgcgtgcg
     3061 cactgccagc ttcagcaccg cggacagtgc cttcgccccc gcctggcggc gcgcgccacc
     3121 gccgcctcag cactgaaggc gcgctgacgt cactcgccgg tcccccgcaa actccccttc
     3181 ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc cggaccgcac cacgcgaggc
     3241 gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg cgccggcgac tcagcgctgc
     3301 ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct gagagcgcag tcagatatcg
     3361 tttaaactcc ggagaagttc ctattctcta gaaagtatag gaacttctcc ggaataactt
     3421 cgtataggat actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag
     3481 caatatgaaa cctcttaata acttcgtata gcatacatta tacgaagtta taacgcgcca
     3541 ctagtgaatt cttacagtct gatctcacct ccactcttat atgattccca tgaaggtatg
     3601 acttttccgc tttgggaagt gactgacaca ttcccccctg tccctccaaa actgcgttgt
     3661 ttcgattctg gtcgattggc tcttctgcac catgtcatta gggaaaatat caacaccagg
     3721 ccaatcatgg cccctgcagt catcaataca tactttcccc agttcgggag acccaggtca
     3781 acccctgaga tctgtttgta cacatcgggg aggtgaactt caacaaaatc ctcagcctca
     3841 tcaccttctt tgaaaactgt agaagggtct gccagggggt gcatcagggg gataactgaa
     3901 gattccaaca actccatatg ttgctggagg agggatgatt gcatctctgg gattaggaca
     3961 tggtcgtcag gccctaatat tataccattg aaaaacaccc cgttcacatg aggatggcac
     4021 cttcctccaa ctttcaaaca cccttttgag gggatgatct cattccaggt ccggactgac
     4081 ttgtagtgag catcagcctc catcaaggtt ttgttgaata tggtatatgc ttttccaaac
     4141 cctgggacaa gttttctcag gtgactgaga cgtctgaaac ttactgactt ggtggtcatg
     4201 atggactcta atgcgtccag acattcctct cttttcttga ctaactcctc cacaacgaga
     4261 tgctcaatct cgtctgagcg aaagtcgtgc aaattcacca actgatctgg agggcaccat
     4321 ttggtctcat ctgatgtttg catcgcgacc catgttccat ccataagtct aagtccaaga
     4381 actccacata acttgagcct gcatgctcct tttagagact tatacaggcc tctttcatcc
     4441 acaaagccgc aagtcttgtt cccgttggat gctctcttcc ctctgctatt ggtaaaaatg
     4501 tcacaaggtg tccttggtct cggattctcg ggcatccaaa tggtgtaatc atggttagtt
     4561 gagcagtagg tagaggacac cgttattcct gagcactttc cgccagggaa gacccttgag
     4621 tgaagggatt tgtcatatgg gtccaaatct gtcacacttg gggatatgat aatgagggac
     4681 tctttggtgg ttcttacagt tcgaagccag tggtagtcgg ggtatggatt gtgtagggac
     4741 tcttcatatc tggggtcacc ggccatcttc cagttatacg cggctctaca tgcgtctggg
     4801 gtggggcgga aatgctttct cttgaatgtg gttgtgacat aaccaacaaa gttggtgtag
     4861 gtctctgcct ctgtcacaac acctgtgcaa gtgaacccgt tcactttgat ggctgagatg
     4921 tatcccactt tgagttccat gtaggagaac tcggacaggt tggtacatcc ttcatcctcc
     4981 acaaccaggt tatttggaca gctgagatgg tgtatgtcaa tagggctcca gggaccaagt
     5041 tcgtctggta tcgtgtaaat ggggaacttc ccgaaacaca acgaaaaacc cagaaggagt
     5101 acaaacaaaa gaacctgagg aaccataggt ccagggttct cctccacgtc tccagcctgc
     5161 ttcagcaggc tgaagttagt agctccgctt cccttgtaca gctcgtccat gccgagagtg
     5221 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
     5281 ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
     5341 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg
     5401 tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg
     5461 ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag
     5521 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg
     5581 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg
     5641 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac
     5701 tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg
     5761 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac
     5821 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg
     5881 gtgaacagct cctcgccctt gctcaccatg ggtccagggt tctcctccac gtctccagcc
     5941 tgcttcagca ggctgaagtt agtagctccg gatccggaga acaagtctgc ctggctcttg
     6001 tcaggcacca gcagctcctt ggatgcgctt tcaagactga agatgtcaga cctgcttttt
     6061 gctttggact tcccccatgc agcgatacca cccacagcta ccaggcagca caggagcact
     6121 gcagtgatca gcatccacat gcggccgtga ttcctggctg gcagagcacg tccaggagca
     6181 ggggcagtgg gagcctctgt gccgttgtcc gtgggcaccg cggggatcgc gctggtcccg
     6241 cagccccact cgtcccgccc gtcgtcgcag tcggggtgtc cgtcgcacag ccagtcctgc
     6301 gggtaacact ccccgtgggc accgggcggc tccgagcagc ggaactgacc gggggggcaa
     6361 cgggacaaag aaccgttacc ggacccgtta ccggtcacgt taccgggcag cagcagcagc
     6421 agcagcgcgg gcagcagccg cgccatggtg gcggcaagct ttactagtgg tggcgctagc
     6481 ataacttcgt ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg
     6541 ttattcttta gaatggtgca aagaataact tcgtataatg tatgctatac gaagttatga
     6601 agttcctatt ctctagaaag tataggaact tcggcgcgcc aatcaacctc tggattacaa
     6661 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     6721 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     6781 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     6841 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     6901 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     6961 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     7021 ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat
     7081 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     7141 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     7201 tcggatctcc ctttgggccg cctccccgcc tggatcctgt gccttctagt tgccagccat
     7261 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     7321 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     7381 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     7441 gggatgcggt gggct
//
