LOCUS       sequence-432184-        6436 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 6436)
  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 6436)
  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-CAG-ConVERGD-eGFP-W3SL
FEATURES             Location/Qualifiers
     source          1..6436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    835..856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(871..876,877..894,895..901)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    909..925
                     /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     914..936
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     933..949
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     933..949
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   989..1129
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1000..1129
                     /label=AAV2 ITR
     primer_bind     1502..1522
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    1626..1673
                     /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      1788..1797
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1794..1796,1797..1799,1800..2513)
                     /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(1794..1796,1797..1799,1800..2510)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1839..1860)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2100..2119)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2447..2468
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2528..2561
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2654..2687
                     /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(2700..2747)
                     /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(2904..2923)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          2931..3947
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(2985..3007)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        4225..4528
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   4614..4743
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     complement(4769..4786)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4769..4785)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4778..4800)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4994..5013)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5113..5135
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5173..5191)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5259..5363
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5364..5432,5433..6224)
                     /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     complement(5582..5601)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6395..6436,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      781 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      841 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      901 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      961 caagctcggc gcgccattgg gatccaatcc tgcaggcagc tgcgcgctcg ctcgctcact
     1021 gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
     1081 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg gagctccggt
     1141 taccggccgc tttaaaaaac ctcccacatc tccccctgaa cctgaaacat aaaatgaatg
     1201 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     1261 tcacaaattt cacaaataaa gctagatggt tacaaataaa gcaatagcat cacaaatttc
     1321 acaaataaac accacggaat tgtcagtgcc caacagccga gcccctgtcc agcagcgggc
     1381 aaggcaggcg gcgatgagtt ccgccgtggc aagaactaac caggatttat acaaggagga
     1441 gaaaatgaaa gccatacggg aagcaatagc atgatacaaa ggcattaaag cagcgtatcc
     1501 acatagcgta aaaggagcaa catagttaag aataccagtc aatctttcac aaattttgta
     1561 atccagaggt tgattatctc gggcatcgaa tatcgaatcg gctaagctct agaaagcttc
     1621 gtacggaagt tactattccg aagttcctat tctctagaaa gtataggaac ttcgtgtcga
     1681 tggtgatgct tggcaattcg ggtcgatggt gaagcattgg tcttcctatt ttgatgaagt
     1741 tcctattctt caaaaggtat aggaacttcc tcgagaccgg tactagtgcc accatggtga
     1801 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1861 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1921 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1981 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     2041 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     2101 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     2161 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     2221 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     2281 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     2341 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2401 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2461 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taagcatgca
     2521 tgaattcata acttcgtata atgtatgcta tacgaagtta ttttctttct ttgcgcgtcc
     2581 tggattcgcg gaaacgcgta catccagctg acgagtccca aataggacga aacgcgcttt
     2641 tctttttgtt tttataactt cgtataatgt atgctatacg aagttatctt aaggtcgacg
     2701 aagttcctat actttctaga gaataggaac ttcggaatag gaacttcgtg tcgatggtga
     2761 tgcttggcaa ttcgggtcga tggtgaagca ttggtcttcc tattttgatg aagttcctat
     2821 accttttgaa gaataggaac ttcgctagcg gtacccccgg gcccgcggta tcaaatcaat
     2881 tctttgccaa aatgatgaga cagcacaata accagcacgt tgcccaggag ctgtaggaaa
     2941 aagaagaagg catgaacatg gttagcagag gctctagagc cgccggtcac acgccagaag
     3001 ccgaaccccg ccctgccccg tcccccccga aggcagccgt ccccccgcgg acagccccga
     3061 ggctggagag ggagaagggg acggcggcgc ggcgacgcac gaaggccctc cccgcccatt
     3121 tccttcctgc cggcgccgca ccgcttcgcc ccgcgcccgc tagagggggt gcggcggcgc
     3181 ctcccagatt tcggctccgc acagatttgg gacaaaggaa gtccctgcgc cctctcgcac
     3241 gattaccata aaaggcaatg gctgcggctc gccgcgcctc gacagccgcc ggcgctccgg
     3301 gggccgccgc gccccycccc cgagcccycc ccggcccgmg gcggccccgc cccgcccggc
     3361 acccccmcct gccgccaccc cccgcccggc acggcgagcc ccgcgccacg ccccgtacgg
     3421 agccccgcac ccgaagccgg gccgtgctca gcaactcggg gaggggggtg cagggggggt
     3481 tgcagcccga ccgacgcgcc cacaccccct gctcaccccc ccacgcacac accccgcacg
     3541 cagcctttgt tcccctcgca gccccccccg caccgcgggg caccgccccc ggccgcgctc
     3601 ccctcgcgca cactgcggag cgcacaaagc cccgcgccgc gcccgcagcg ctcacagccg
     3661 ccgggcagcg cggagccgca cgcggcgctc cccacgcaca cacacacgca cgcacccccc
     3721 gagccgctcc ccccgcacaa agggccctcc cggagcccct caaggctttc acgcagccac
     3781 agaaaagaaa caagccgtca ttaaaccaag cgctaattac agcccggagg agaagggccg
     3841 tcccgcccgc tcacctgtgg gagtaacgcg gtcagtcaga gccggggcgg gcggcgcgag
     3901 gcggcggcgg agcggggcac ggggcgaagg cagcgcgcag cgactcccgc ccgccgcgcg
     3961 cttcgctttt tatagggccg ccgccgccgc cgcctcgcca taaaaggaaa ctttcggagc
     4021 gcgccgctct gattggctgc cgccgcacct ctccgccycs ccccsccccs ccccycsccc
     4081 cscccccccc ccccyggcgc gcsccccccc cccccccgcc cccatcgctg cacaaaataa
     4141 ttaaaaaata aataaataca aaattggggg tggggagggg ggggagatgg ggagagtgaa
     4201 gcagaacgtg gggctcacct cgaccatggt aatagcgatg actaatacgt agatgtactg
     4261 ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc
     4321 gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg
     4381 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac
     4441 tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg
     4501 cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga
     4561 ccccgtaatt gattactatt aataactaga ttcctgcaga cgcgtgcggc cgcaggaacc
     4621 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4681 accaaaggtc gcccgacgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     4741 aggattggat cccaatacgc gtcaattcac tggccgtcgt tttacaacgt cgtgactggg
     4801 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     4861 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     4921 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     4981 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     5041 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5101 ctgtgacagt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5161 cgagacaaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     5221 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5281 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5341 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5401 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5461 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5521 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5581 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5641 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5701 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5761 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5821 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5881 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     5941 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6001 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6061 ctggagccgg tgagcgtggt tctcgcggta tcattgcagc actggggcca gatggtaagc
     6121 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6181 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6241 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6301 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6361 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6421 tctgctgctt gcaaac
//
