LOCUS       sequence-432139-        5341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides AND intersectional (Cre+Flp+vCre+Vika) expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5341)
  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 5341)
  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-ConFonvConNon-eGFP-W3SL
FEATURES             Location/Qualifiers
     source          1..5341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(105..124)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      918..1506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1407..1426
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1794..1815
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1830..1835,1836..1853,1854..1860)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1868..1884
                     /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)."
     primer_bind     1873..1895
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1892..1908
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1892..1908
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1948..2088
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1959..2088
                     /label=AAV2 ITR
     primer_bind     2461..2481
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(2906..2939)
                     /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(3032..3065)
                     /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    3066..3099
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3192..3225
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(3241..3954,3955..3957,3958..3960))
                     /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(3244..3954,3955..3957,3958..3960))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3286..3307)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3635..3654
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3894..3915
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3957..3966
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3997..4444)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   4478..4607
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     complement(4633..4650)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4633..4649)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4642..4664)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4858..4877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4977..4999
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5037..5055)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5123..5227
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5228..5296,5297..5341,1..747)
                     /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 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
       61 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      121 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
      181 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
      241 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
      301 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
      361 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
      421 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
      481 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
      541 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
      601 ggttctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
      661 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
      721 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
      781 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
      841 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
      901 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
      961 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1021 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     1081 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1141 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1201 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1261 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1321 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1381 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     1441 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1501 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     1561 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     1621 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     1681 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     1741 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     1801 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     1861 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     1921 aagctcggcg cgccattggg atccaatcct gcaggcagct gcgcgctcgc tcgctcactg
     1981 aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     2041 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgg agctccggtt
     2101 accggccgct ttaaaaaacc tcccacatct ccccctgaac ctgaaacata aaatgaatgc
     2161 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2221 cacaaatttc acaaataaag ctagatggtt acaaataaag caatagcatc acaaatttca
     2281 caaataaaca ccacggaatt gtcagtgccc aacagccgag cccctgtcca gcagcgggca
     2341 aggcaggcgg cgatgagttc cgccgtggca agaactaacc aggatttata caaggaggag
     2401 aaaatgaaag ccatacggga agcaatagca tgatacaaag gcattaaagc agcgtatcca
     2461 catagcgtaa aaggagcaac atagttaaga ataccagtca atctttcaca aattttgtaa
     2521 tccagaggtt gattatctcg ggcatcgaat atcgaatcgg ctaagctcta gaaagcttgc
     2581 gtacgtgaat gtcctataag tgtaattata ggacattcaa aaaacaaaaa gaaaagcgcg
     2641 tttcgtccta tttgggactc gtcagctgga tgtacgcgtt tccgcgaatc caggacgcgc
     2701 aaagaaagaa atgaatgtcc tataagtgta attataggac attcatcaat ttccgagaat
     2761 gacagttctc agaaattgaa aaaacaaaaa gaaaagcgcg tttcgtccta tttgggactc
     2821 gtcagctgga tgtacgcgtt tccgcgaatc caggacgcgc aaagaaagaa atcaatttcc
     2881 gagaatgaca gttctcagaa attgagaagt tcctatactt tctagagaat aggaacttca
     2941 aaaacaaaaa gaaaagcgcg tttcgtccta tttgggactc gtcagctgga tgtacgcgtt
     3001 tccgcgaatc caggacgcgc aaagaaagaa agaagttcct atactttcta gagaatagga
     3061 acttcataac ttcgtataat gtatgctata cgaagttata aaaacaaaaa gaaaagcgcg
     3121 tttcgtccta tttgggactc gtcagctgga tgtacgcgtt tccgcgaatc caggacgcgc
     3181 aaagaaagaa aataacttcg tataatgtat gctatacgaa gttatagcat gcatgaattc
     3241 ttacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag
     3301 gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta
     3361 gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc
     3421 ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc
     3481 gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt
     3541 gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac
     3601 cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa
     3661 gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg
     3721 cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag
     3781 ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc
     3841 gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc
     3901 gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat
     3961 ggtggcctcg agaccggtac tagtgctagc ggtaccctgc gctctcaggc acgacacgac
     4021 tcctccgctg cccaccgcag actgaggcag cgctgagtcg ccggcgccgc agcgcagatg
     4081 gtcgcgcccg tgccccccta tctcgcgcct cgcgtggtgc ggtccggctg ggccggcggc
     4141 ggcgcggacg cgaccaaggt ggccgggaag gggagtttgc gggggaccgg cgagtgacgt
     4201 cagcgcgcct tcagtgctga ggcggcggtg gcgcgcgccg ccaggcgggg gcgaaggcac
     4261 tgtccgcggt gctgaagctg gcagtgcgca cgcgcctcgc cgcatcctgt ttcccctccc
     4321 cctctctgat aggggatgcg caatttgggg aatgggggtt gggtgcttgt ccagtgggtc
     4381 ggggtcggtc gtcaggtagg cacccccacc ccgcctcatc ctggtcctaa aacccacttg
     4441 cactgcgatc gcgggcccgt gcagcatctg cggccgcagg aacccctagt gatggagttg
     4501 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4561 cgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggatt ggatcccaat
     4621 acgcgtcaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     4681 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     4741 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     4801 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac
     4861 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     4921 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga cagtctccgg
     4981 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac aaaagggcct
     5041 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5101 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5161 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5221 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5281 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5341 g
//
