LOCUS       sequence-432141-        5209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides AND intersectional (Cre+Flp+vCre) expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5209)
  AUTHORS   Hughes AC, Pollard BG, Xu B, Gammons JW, Chapman P, Bikoff JB, 
            Schwarz LA
  TITLE     A Novel Single Vector Intersectional AAV Strategy for Interrogating 
            Cellular Diversity and Brain Function.
  JOURNAL   bioRxiv. 2023 Feb 8:2023.02.07.527312. doi: 
            10.1101/2023.02.07.527312. Preprint.
  PUBMED    36798174
REFERENCE   2  (bases 1 to 5209)
  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: "bioRxiv. 
            2023 Feb 8:2023.02.07.527312. doi: 10.1101/2023.02.07.527312. 
            Preprint."
COMMENT     Sequence Label: pAAV-hSyn-ConVERGD-ConFonvCon-eGFP-W3SL
FEATURES             Location/Qualifiers
     source          1..5209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(260..364)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     432..450
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(488..510)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     610..629
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     823..845
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     837..854
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     838..854
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   880..1009
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        1043..1490
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1521..1530
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1527..1529,1530..1532,1533..2246)
                     /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             join(1527..1529,1530..1532,1533..2243)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1572..1593)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1833..1852)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2180..2201
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2262..2295)
                     /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(2388..2421)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2422..2469
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    2562..2609
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     complement(2874..2894)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3267..3407
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3267..3396
                     /label=AAV2 ITR
     primer_bind     complement(3447..3463)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3447..3463)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3460..3482)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3471..3487
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3495..3501,3502..3519,3520..3525))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3540..3561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3849..4437)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3929..3948)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4608..5209,1..190,191..259))
                     /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"
ORIGIN
        1 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
       61 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
      121 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
      181 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
      241 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
      301 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
      361 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
      421 taacctataa aaataggcgt atcacgaggc ccttttgtct cgcgcgtttc ggtgatgacg
      481 gtgaaaacct ctgacacatg cagctcccgg agactgtcac agcttgtctg taagcggatg
      541 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
      601 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
      661 cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca
      721 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      781 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      841 aaacgacggc cagtgaattg acgcgtattg ggatccaatc ctgcaggcag ctgcgcgctc
      901 gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
      961 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcaga
     1021 tgctgcacgg gcccgcgatc gcagtgcaag tgggttttag gaccaggatg aggcggggtg
     1081 ggggtgccta cctgacgacc gaccccgacc cactggacaa gcacccaacc cccattcccc
     1141 aaattgcgca tcccctatca gagaggggga ggggaaacag gatgcggcga ggcgcgtgcg
     1201 cactgccagc ttcagcaccg cggacagtgc cttcgccccc gcctggcggc gcgcgccacc
     1261 gccgcctcag cactgaaggc gcgctgacgt cactcgccgg tcccccgcaa actccccttc
     1321 ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc cggaccgcac cacgcgaggc
     1381 gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg cgccggcgac tcagcgctgc
     1441 ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct gagagcgcag ggtaccgcta
     1501 gcactagtac cggtctcgag gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     1561 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1621 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1681 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1741 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1801 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1861 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1921 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1981 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2041 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2101 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     2161 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2221 tcggcatgga cgagctgtac aagtaagaat tcatgcatgc tataacttcg tatagcatac
     2281 attatacgaa gttattttct ttctttgcgc gtcctggatt cgcggaaacg cgtacatcca
     2341 gctgacgagt cccaaatagg acgaaacgcg cttttctttt tgtttttata acttcgtata
     2401 gcatacatta tacgaagtta tgaagttcct attccgaagt tcctattctc tagaaagtat
     2461 aggaacttct ttctttcttt gcgcgtcctg gattcgcgga aacgcgtaca tccagctgac
     2521 gagtcccaaa taggacgaaa cgcgcttttc tttttgtttt tgaagttact attccgaagt
     2581 tcctattctc tagaaagtat aggaacttct caatttctga gaactgtcat tctcggaaat
     2641 tgatttcttt ctttgcgcgt cctggattcg cggaaacgcg tacatccagc tgacgagtcc
     2701 caaataggac gaaacgcgct tttctttttg ttttttcaat ttctgagaac tgtcattctc
     2761 ggaaattgac gtacgcaagc tttctagagc ttagccgatt cgatattcga tgcccgagat
     2821 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     2881 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     2941 atggctttca ttttctcctc cttgtataaa tcctggttag ttcttgccac ggcggaactc
     3001 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3061 gtggtgttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat ctagctttat
     3121 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     3181 taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt
     3241 ttaaagcggc cggtaaccgg agctccagga acccctagtg atggagttgg ccactccctc
     3301 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     3361 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggatt ggatcccaat
     3421 ggcgcgccga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3481 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3541 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3601 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3661 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3721 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3781 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3841 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3901 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3961 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4021 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4081 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4141 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4201 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4261 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4321 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4381 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4441 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4501 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4561 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4621 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4681 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4741 ataccgcgag aaccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4801 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4861 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4921 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4981 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5041 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5101 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5161 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgct
//
