LOCUS       sequence-432143-        5275 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides NOT intersectional (Flp NOT Cre) expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5275)
  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 5275)
  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-CoffFon-eGFP-W3SL
FEATURES             Location/Qualifiers
     source          1..5275
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   14..143
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        177..624
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    637..670
                     /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(767..800)
                     /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(851..884)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    977..1010
                     /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    1061..1094
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     CDS             complement(join(1109..1822,1823..1825,1826..1828))
                     /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(1112..1822,1823..1825,1826..1828))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1154..1175)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1503..1522
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1762..1783
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1825..1834
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1853..1886)
                     /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)"
     primer_bind     complement(2074..2094)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2467..2607
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2467..2596
                     /label=AAV2 ITR
     primer_bind     complement(2647..2663)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2647..2663)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2660..2682)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2671..2687
                     /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(2695..2701,2702..2719,2720..2725))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2740..2761
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3049..3637)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3129..3148)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3808..4599,4600..4668))
                     /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     4431..4450
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4669..4773)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4841..4859
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4897..4919)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5019..5038
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5232..5254
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5246..5263
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5247..5263
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 attgggatcc aatcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg
       61 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc
      121 caactccatc actaggggtt cctgcggccg cagatgctgc acgggcccgc gatcgcagtg
      181 caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac gaccgacccc
      241 gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg
      301 gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca
      361 gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg
      421 acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc
      481 gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac
      541 catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggagga
      601 gtcgtgtcgt gcctgagagc gcagggtacc gctagcgaag ttcctattct ctagaaagta
      661 taggaacttc gcagaatggt agctggattg tagctgctat tagcaatatg aaacctctta
      721 gaagttccta ttcttcaaaa ggtataggaa cttcgtcgac cttaagataa cttcgtatag
      781 gatactttat acgaagttat gtagctggag caatttgaaa agattgttgt attacctcta
      841 gctgcgctta ataacttcgt atagtatacc ttatacgaag ttattttctt tctttgcgcg
      901 tcctggattc gcggaaacgc gtacatccag ctgacgagtc ccaaatagga cgaaacgcgc
      961 ttttcttttt gtttttataa cttcgtataa agtatcctat acgaagttat ttgccttaac
     1021 ccagaaatta tcactgttat tctttagata ggtgcaaaga ataacttcgt ataaggtata
     1081 ctatacgaag ttatgaattc atgcatgctt acttgtacag ctcgtccatg ccgagagtga
     1141 tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt
     1201 tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc
     1261 cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt
     1321 ggcggatctt gaagttcacc ttgatgccgt tcttctgctt gtcggccatg atatagacgt
     1381 tgtggctgtt gtagttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt
     1441 cgatgccctt cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc
     1501 gggtcttgta gttgccgtcg tccttgaaga agatggtgcg ctcctggacg tagccttcgg
     1561 gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact
     1621 gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg
     1681 tgcagatgaa cttcagggtc agcttgccgt aggtggcatc gccctcgccc tcgccggaca
     1741 cgctgaactt gtggccgttt acgtcgccgt ccagctcgac caggatgggc accaccccgg
     1801 tgaacagctc ctcgcccttg ctcaccatgg tggcactagt accggtctcg aggaagttcc
     1861 tatactttct agagaatagg aacttctaag cagacaatct tgtaatacat cagcagctag
     1921 tagattgctc cagctagaag ttcctatacc ttttgaagaa taggaacttc cgtacgaagc
     1981 tttctagagc ttagccgatt cgatattcga tgcccgagat aatcaacctc tggattacaa
     2041 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     2101 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     2161 cttgtataaa tcctggttag ttcttgccac ggcggaactc atcgccgcct gccttgcccg
     2221 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttta tttgtgaaat
     2281 ttgtgatgct attgctttat ttgtaaccat ctagctttat ttgtgaaatt tgtgatgcta
     2341 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     2401 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcggc cggtaaccgg
     2461 agctccagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     2521 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     2581 gcgagcgagc gcgcagctgc ctgcaggatt ggatcccaat ggcgcgccga gcttggcgta
     2641 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2701 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2761 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2821 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2881 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2941 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3001 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3061 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3121 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3181 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3241 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3301 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3361 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3421 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3481 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3541 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3601 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3661 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3721 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3781 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3841 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3901 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag aaccacgctc
     3961 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4021 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4081 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4141 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4201 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4261 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4321 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4381 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4441 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4501 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4561 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4621 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4681 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4741 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4801 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     4861 cttttgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     4921 gactgtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     4981 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     5041 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     5101 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     5161 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     5221 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattga cgcgt
//
