LOCUS       sequence-432190-        7085 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 7085)
  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 7085)
  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-WPRE
FEATURES             Location/Qualifiers
     source          1..7085
                     /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
     polyA_signal    2108..2584
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     2366..2385
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    2608..3196
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             2714..2725
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3123..3143
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    3226..3273
                     /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      3388..3397
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3394..3396,3397..3399,3400..4113)
                     /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(3394..3396,3397..3399,3400..4110)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3439..3460)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3700..3719)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4047..4068
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    4128..4161
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4254..4287
                     /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(4300..4347)
                     /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(4504..4523)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          4531..5547
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(4585..4607)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(5548..5824)
                     /label=chicken beta-actin promoter
     enhancer        5826..6129
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   6215..6344
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     complement(6370..6387)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6370..6386)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6379..6401)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6595..6614)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6714..6736
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6774..6792)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6860..6964
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6965..7033,7034..7085,1..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 gttacctaca
     2101 aaatcagaag gacagggaag ggagcagtgg ttcacgcctg taatcccagc aatttgggag
     2161 gccaaggtgg gtagatcacc tgagattagg agttggagac cagcctggcc aatatggtga
     2221 aaccccgtct ctaccaaaaa aacaaaaatt agctgagcct ggtcatgcat gcctggaatc
     2281 ccaacaactc gggaggctga ggcaggagaa tcgcttgaac ccaggaggcg gagattgcag
     2341 tgagccaaga ttgtgccact gcactccagc ttggttccca atagaccccg caggccctac
     2401 aggttgtctt cccaacttgc cccttgctcc ataccacccc cctccacccc ataatattat
     2461 agaaggacac ctagtcagac aaaatgatgc aacttaattt tattaggaca aggctggtgg
     2521 gcactggagt ggcaacttcc agggccagga gaggcactgg ggaggggtca cagggatgcc
     2581 acccgtagat cgatctacgg tatcgatgcg gggaggcggc ccaaagggag atccgactcg
     2641 tctgagggcg aaggcgaaga cgcggaagag gccgcagagc cggcagcagg ccgcgggaag
     2701 gaaggtccgc tggattgagg gccgaaggga cgtagcagaa ggacgtcccg cgcagaatcc
     2761 aggtggcaac acaggcgagc agccaaggaa aggacgatga tttccccgac aacaccacgg
     2821 aattgtcagt gcccaacagc cgagcccctg tccagcagcg ggcaaggcag gcggcgatga
     2881 gttccgccgt ggcaataggg agggggaaag cgaaagtccc ggaaaggagc tgacaggtgg
     2941 tggcaatgcc ccaaccagtg ggggttgcgt cagcaaacac agtgcacacc acgccacgtt
     3001 gcctgacaac gggccacaac tcctcataaa gagacagcaa ccaggattta tacaaggagg
     3061 agaaaatgaa agccatacgg gaagcaatag catgatacaa aggcattaaa gcagcgtatc
     3121 cacatagcgt aaaaggagca acatagttaa gaataccagt caatctttca caaattttgt
     3181 aatccagagg ttgattatcg atcggccgca tataagcttc gtacggaagt tactattccg
     3241 aagttcctat tctctagaaa gtataggaac ttcgtgtcga tggtgatgct tggcaattcg
     3301 ggtcgatggt gaagcattgg tcttcctatt ttgatgaagt tcctattctt caaaaggtat
     3361 aggaacttcc tcgagaccgg tactagtgcc accatggtga gcaagggcga ggagctgttc
     3421 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3481 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3541 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     3601 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3661 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3721 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3781 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     3841 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     3901 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3961 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     4021 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     4081 atcactctcg gcatggacga gctgtacaag taagcatgca tgaattcata acttcgtata
     4141 atgtatgcta tacgaagtta ttttctttct ttgcgcgtcc tggattcgcg gaaacgcgta
     4201 catccagctg acgagtccca aataggacga aacgcgcttt tctttttgtt tttataactt
     4261 cgtataatgt atgctatacg aagttatctt aaggtcgacg aagttcctat actttctaga
     4321 gaataggaac ttcggaatag gaacttcgtg tcgatggtga tgcttggcaa ttcgggtcga
     4381 tggtgaagca ttggtcttcc tattttgatg aagttcctat accttttgaa gaataggaac
     4441 ttcgctagcg gtacccccgg gcccgcggta tcaaatcaat tctttgccaa aatgatgaga
     4501 cagcacaata accagcacgt tgcccaggag ctgtaggaaa aagaagaagg catgaacatg
     4561 gttagcagag gctctagagc cgccggtcac acgccagaag ccgaaccccg ccctgccccg
     4621 tcccccccga aggcagccgt ccccccgcgg acagccccga ggctggagag ggagaagggg
     4681 acggcggcgc ggcgacgcac gaaggccctc cccgcccatt tccttcctgc cggcgccgca
     4741 ccgcttcgcc ccgcgcccgc tagagggggt gcggcggcgc ctcccagatt tcggctccgc
     4801 acagatttgg gacaaaggaa gtccctgcgc cctctcgcac gattaccata aaaggcaatg
     4861 gctgcggctc gccgcgcctc gacagccgcc ggcgctccgg gggccgccgc gcccctcccc
     4921 cgagccctcc ccggcccgag gcggccccgc cccgcccggc acccccacct gccgccaccc
     4981 cccgcccggc acggcgagcc ccgcgccacg ccccgtacgg agccccgcac ccgaagccgg
     5041 gccgtgctca gcaactcggg gaggggggtg cagggggggt tgcagcccga ccgacgcgcc
     5101 cacaccccct gctcaccccc ccacgcacac accccgcacg cagcctttgt tcccctcgca
     5161 gccccccccg caccgcgggg caccgccccc ggccgcgctc ccctcgcgca cactgcggag
     5221 cgcacaaagc cccgcgccgc gcccgcagcg ctcacagccg ccgggcagcg cggagccgca
     5281 cgcggcgctc cccacgcaca cacacacgca cgcacccccc gagccgctcc ccccgcacaa
     5341 agggccctcc cggagcccct caaggctttc acgcagccac agaaaagaaa caagccgtca
     5401 ttaaaccaag cgctaattac agcccggagg agaagggccg tcccgcccgc tcacctgtgg
     5461 gagtaacgcg gtcagtcaga gccggggcgg gcggcgcgag gcggcggcgg agcggggcac
     5521 ggggcgaagg cagcgcgcag cgactcccgc ccgccgcgcg cttcgctttt tatagggccg
     5581 ccgccgccgc cgcctcgcca taaaaggaaa ctttcggagc gcgccgctct gattggctgc
     5641 cgccgcacct ctccgcctcg ccccgccccg cccctcgccc cgccccgccc cgcctggcgc
     5701 gcgccccccc ccccccccgc ccccatcgct gcacaaaata attaaaaaat aaataaatac
     5761 aaaattgggg gtggggaggg gggggagatg gggagagtga agcagaacgt ggggctcacc
     5821 tcgaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat
     5881 aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg
     5941 ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac
     6001 tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt
     6061 attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt
     6121 tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat
     6181 taataactag attcctgcag acgcgtgcgg ccgcaggaac ccctagtgat ggagttggcc
     6241 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     6301 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggattgga tcccaatacg
     6361 cgtcaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     6421 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     6481 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     6541 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     6601 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     6661 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgacag tctccgggag
     6721 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacaaa agggcctcgt
     6781 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     6841 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6901 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     6961 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     7021 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     7081 tgcac
//
