LOCUS       sequence-432121-        5204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides AND intersectional (Cre+Flp) expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5204)
  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 5204)
  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-nEF-ConVERGD-eGFP-W3SL
FEATURES             Location/Qualifiers
     source          1..5204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(98..117)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      911..1499
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1400..1419
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1787..1808
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1823..1828,1829..1846,1847..1853)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1861..1877
                     /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     1866..1888
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1885..1901
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1885..1901
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1941..2081
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1952..2081
                     /label=AAV2 ITR
     primer_bind     2454..2474
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    2578..2625
                     /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)."
     regulatory      2740..2749
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2746..2748,2749..2751,2752..3465)
                     /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(2746..2748,2749..2751,2752..3462)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2791..2812)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3052..3071)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3399..3420
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3480..3513
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3606..3639
                     /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(3652..3699)
                     /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)."
     LTR             3808..4076
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     promoter        complement(4089..4300)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     repeat_region   4334..4463
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     complement(4489..4506)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4489..4505)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4498..4520)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4714..4733)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4833..4855
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4893..4911)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4979..5083
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5084..5152,5153..5204,1..740)
                     /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 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
       61 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
      121 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
      181 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
      241 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
      301 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
      361 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
      421 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
      481 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
      541 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttctc
      601 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
      661 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
      721 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
      781 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
      841 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
      901 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
      961 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1021 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     1081 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1141 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     1201 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     1261 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     1321 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     1381 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     1441 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     1501 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     1561 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     1621 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     1681 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     1741 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     1801 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     1861 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctcg
     1921 gcgcgccatt gggatccaat cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc
     1981 ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
     2041 gcgcagagag ggagtggcca actccatcac taggggttcc tggagctccg gttaccggcc
     2101 gctttaaaaa acctcccaca tctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2161 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2221 ttcacaaata aagctagatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     2281 acaccacgga attgtcagtg cccaacagcc gagcccctgt ccagcagcgg gcaaggcagg
     2341 cggcgatgag ttccgccgtg gcaagaacta accaggattt atacaaggag gagaaaatga
     2401 aagccatacg ggaagcaata gcatgataca aaggcattaa agcagcgtat ccacatagcg
     2461 taaaaggagc aacatagtta agaataccag tcaatctttc acaaattttg taatccagag
     2521 gttgattatc tcgggcatcg aatatcgaat cggctaagct ctagaaagct tcgtacggaa
     2581 gttactattc cgaagttcct attctctaga aagtatagga acttcgtgtc gatggtgatg
     2641 cttggcaatt cgggtcgatg gtgaagcatt ggtcttccta ttttgatgaa gttcctattc
     2701 ttcaaaaggt ataggaactt cctcgagacc ggtactagtg ccaccatggt gagcaagggc
     2761 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     2821 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     2881 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     2941 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     3001 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     3061 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     3121 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     3181 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     3241 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     3301 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     3361 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     3421 accgccgccg ggatcactct cggcatggac gagctgtaca agtaagcatg catgaattca
     3481 taacttcgta taatgtatgc tatacgaagt tattttcttt ctttgcgcgt cctggattcg
     3541 cggaaacgcg tacatccagc tgacgagtcc caaataggac gaaacgcgct tttctttttg
     3601 tttttataac ttcgtataat gtatgctata cgaagttatc ttaaggtcga cgaagttcct
     3661 atactttcta gagaatagga acttcggaat aggaacttcg tgtcgatggt gatgcttggc
     3721 aattcgggtc gatggtgaag cattggtctt cctattttga tgaagttcct ataccttttg
     3781 aagaatagga acttcgctag cggtaccgat ctgtaacggc gcagaacaga aaacgaaaca
     3841 aagacgtaga gttgagcaag cagggtcagg caaagcgtgg agagccggct gagtctaggt
     3901 aggctccaag ggagcgccgg acaaaggccc ggtctcgacc tgagctttaa acttacctag
     3961 acggcggacg cagttcagga ggcaccacag gcgggaggcg gcagaacgcg actcaaccgg
     4021 cgtggatggc ggcctcaggt agggcggcgg gcgcgtgaag gagagatgcg agcccctcga
     4081 agcttcagct gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact
     4141 gcacttatat acggttctcc cccaccctcg ggaaaaaggc ggagccagta cacgacatca
     4201 ctttcccagt ttaccccgcg ccaccttctc taggcacccg ttcaattgcc gacccctccc
     4261 cccaacttct cggggactgt gggcgatgtg cgctctgccc gcgatcgcgg gcccgtgcag
     4321 catctgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4381 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct cagtgagcga
     4441 gcgagcgcgc agctgcctgc aggattggat cccaatacgc gtcaattcac tggccgtcgt
     4501 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     4561 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     4621 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     4681 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4741 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4801 ggcatccgct tacagacaag ctgtgacagt ctccgggagc tgcatgtgtc agaggttttc
     4861 accgtcatca ccgaaacgcg cgagacaaaa gggcctcgtg atacgcctat ttttataggt
     4921 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     4981 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5041 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5101 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5161 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcac
//
