LOCUS       sequence-432135-        5852 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 5852)
  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 5852)
  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-WPRE
FEATURES             Location/Qualifiers
     source          1..5852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(79..870,871..939))
                     /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     702..721
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(940..1044)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1112..1130
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1168..1190)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1290..1309
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1503..1525
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1517..1534
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1518..1534
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1560..1689
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        1723..1934
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1947..2215
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    2324..2371
                     /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(2384..2417)
                     /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(2510..2543)
                     /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(2558..3271,3272..3274,3275..3277))
                     /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(2561..3271,3272..3274,3275..3277))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2603..2624)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2952..2971
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3211..3232
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3274..3283
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3398..3445)
                     /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)."
     misc_feature    3475..4063
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3528..3548)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3946..3957)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4087..4563
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4286..4305)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4590..4730
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4590..4719
                     /label=AAV2 ITR
     primer_bind     complement(4770..4786)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4770..4786)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4783..4805)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4794..4810
                     /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(4818..4824,4825..4842,4843..4848))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4863..4884
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5172..5760)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5252..5271)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
       61 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
      121 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
      181 gggcttacca tctggcccca gtgctgcaat gataccgcga gaaccacgct caccggctcc
      241 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
      301 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
      361 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
      421 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
      481 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
      541 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
      601 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
      661 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
      721 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
      781 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
      841 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
      901 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
      961 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     1021 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     1081 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc ccttttgtct
     1141 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agactgtcac
     1201 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     1261 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     1321 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
     1381 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     1441 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     1501 ttcccagtca cgacgttgta aaacgacggc cagtgaattg acgcgtattg ggatccaatc
     1561 ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg
     1621 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
     1681 ggggttcctg cggccgcaga tgctgcacgg gcccgcgatc gcgggcagag cgcacatcgc
     1741 ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt
     1801 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     1861 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     1921 ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc
     1981 ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg
     2041 cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt
     2101 gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc
     2161 tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac agatcggtac
     2221 cgctagcgaa gttcctattc ttcaaaaggt ataggaactt catcaaaata ggaagaccaa
     2281 tgcttcacca tcgacccgaa ttgccaagca tcaccatcga cacgaagttc ctattccgaa
     2341 gttcctattc tctagaaagt ataggaactt cgtcgacctt aagataactt cgtatagcat
     2401 acattatacg aagttataaa aacaaaaaga aaagcgcgtt tcgtcctatt tgggactcgt
     2461 cagctggatg tacgcgtttc cgcgaatcca ggacgcgcaa agaaagaaaa taacttcgta
     2521 tagcatacat tatacgaagt tatgaattca tgcatgctta cttgtacagc tcgtccatgc
     2581 cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt
     2641 tggggtcttt gctcagggcg gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg
     2701 ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc gagctgcacg ctgccgtcct
     2761 cgatgttgtg gcggatcttg aagttcacct tgatgccgtt cttctgcttg tcggccatga
     2821 tatagacgtt gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct
     2881 ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca
     2941 cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt
     3001 agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc
     3061 tgaagcactg cacgccgtag gtcagggtgg tcacgagggt gggccagggc acgggcagct
     3121 tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcgccct
     3181 cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca
     3241 ccaccccggt gaacagctcc tcgcccttgc tcaccatggt ggcactagta ccggtctcga
     3301 ggaagttcct ataccttttg aagaatagga acttcatcaa aataggaaga ccaatgcttc
     3361 accatcgacc cgaattgcca agcatcacca tcgacacgaa gttcctatac tttctagaga
     3421 ataggaactt cggaatagta acttccgtac gaagcttata tgcggccgat cgataatcaa
     3481 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3541 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3601 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3661 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3721 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3781 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     3841 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     3901 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     3961 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     4021 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtagatcg
     4081 atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc
     4141 actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag
     4201 gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg
     4261 aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa
     4321 tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc
     4381 gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag
     4441 agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac
     4501 ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc
     4561 cttctgattt tgtaggtaac cggagctcca ggaaccccta gtgatggagt tggccactcc
     4621 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4681 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg attggatccc
     4741 aatggcgcgc cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4801 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4861 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4921 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4981 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5041 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5101 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5161 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5221 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5281 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5341 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5401 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5461 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5521 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5581 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5641 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5701 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5761 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5821 gggattttgg tcatgagatt atcaaaaagg at
//
