LOCUS       sequence-432133-        5957 bp    DNA     circular SYN 14-APR-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 5957)
  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 5957)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-Ef1a-ConVERGD-eGFP-W3SL
FEATURES             Location/Qualifiers
     source          1..5957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(82..873,874..942))
                     /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     705..724
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(943..1047)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1115..1133
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1171..1193)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1293..1312
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1506..1528
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1520..1537
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1521..1537
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1563..1692
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        1793..2971
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2024..2962
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2919..2939
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    3080..3127
                     /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    complement(3140..3173)
                     /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(3266..3299)
                     /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(3314..4027,4028..4030,4031..4033))
                     /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(3317..4027,4028..4030,4031..4033))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3359..3380)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3708..3727
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3967..3988
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4030..4039
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(4154..4201)
                     /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(4305..4325)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4698..4838
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4698..4827
                     /label=AAV2 ITR
     primer_bind     complement(4878..4894)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4878..4894)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4891..4913)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4902..4918
                     /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(4926..4932,4933..4950,4951..4956))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4971..4992
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5280..5868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5360..5379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
       61 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
      121 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
      181 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagaaccac gctcaccggc
      241 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
      301 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
      361 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
      421 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
      481 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
      541 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
      601 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
      661 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
      721 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
      781 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
      841 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
      901 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
      961 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1021 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     1081 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggcccttttg
     1141 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagactgt
     1201 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     1261 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     1321 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     1381 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     1441 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     1501 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgacgcgta ttgggatcca
     1561 atcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct
     1621 ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca
     1681 ctaggggttc ctgcggccgc acgcgtaagc tttgcaaaga tggataaagt tttaaacaga
     1741 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg
     1801 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     1861 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     1921 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     1981 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg
     2041 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac
     2101 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc
     2161 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg
     2221 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa
     2281 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag
     2341 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg
     2401 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc
     2461 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc
     2521 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg
     2581 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg
     2641 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc
     2701 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc
     2761 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc
     2821 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt
     2881 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca
     2941 aagttttttt cttccatttc aggtgtcgtg aggtaccgct agcgaagttc ctattcttca
     3001 aaaggtatag gaacttcatc aaaataggaa gaccaatgct tcaccatcga cccgaattgc
     3061 caagcatcac catcgacacg aagttcctat tccgaagttc ctattctcta gaaagtatag
     3121 gaacttcgtc gaccttaaga taacttcgta tagcatacat tatacgaagt tataaaaaca
     3181 aaaagaaaag cgcgtttcgt cctatttggg actcgtcagc tggatgtacg cgtttccgcg
     3241 aatccaggac gcgcaaagaa agaaaataac ttcgtatagc atacattata cgaagttatg
     3301 aattcatgca tgcttacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     3361 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact
     3421 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     3481 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt
     3541 tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt
     3601 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
     3661 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc
     3721 cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
     3781 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca
     3841 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca
     3901 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
     3961 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     4021 ccttgctcac catggtggca ctagtaccgg tctcgaggaa gttcctatac cttttgaaga
     4081 ataggaactt catcaaaata ggaagaccaa tgcttcacca tcgacccgaa ttgccaagca
     4141 tcaccatcga cacgaagttc ctatactttc tagagaatag gaacttcgga atagtaactt
     4201 ccgtacgaag ctttctagag cttagccgat tcgatattcg atgcccgaga taatcaacct
     4261 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     4321 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     4381 attttctcct ccttgtataa atcctggtta gttcttgcca cggcggaact catcgccgcc
     4441 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttt
     4501 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca tctagcttta tttgtgaaat
     4561 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     4621 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcgg
     4681 ccggtaaccg gagctccagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4741 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4801 ggcctcagtg agcgagcgag cgcgcagctg cctgcaggat tggatcccaa tggcgcgccg
     4861 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4921 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4981 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5041 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5101 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5161 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5221 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5281 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5341 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5401 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5461 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5521 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5581 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5641 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5701 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5761 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5821 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5881 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5941 atgagattat caaaaag
//
