LOCUS       sequence-432148-        6605 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 6605)
  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 6605)
  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-Ef1a-ConVERGD-eGFP-WPRE
FEATURES             Location/Qualifiers
     source          1..6605
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    103..124
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(139..144,145..162,163..169)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    177..193
                     /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     182..204
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     201..217
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     201..217
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   257..397
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   268..397
                     /label=AAV2 ITR
     polyA_signal    424..900
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     682..701
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    924..1512
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1030..1041
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1439..1459
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    1542..1589
                     /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      1704..1713
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1710..1712,1713..1715,1716..2429)
                     /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(1710..1712,1713..1715,1716..2426)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1755..1776)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2016..2035)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2363..2384
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2444..2477
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2570..2603
                     /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(2616..2663)
                     /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)."
     promoter        complement(2772..3950)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2781..3719
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2804..2824)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   4051..4180
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     complement(4206..4223)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4206..4222)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4215..4237)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4431..4450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4550..4572
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4610..4628)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4696..4800
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4801..4869,4870..5661)
                     /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(5019..5038)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5832..6420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6321..6340
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
       61 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
      121 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
      181 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctcggcgc
      241 gccattggga tccaatcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg
      301 gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc
      361 agagagggag tggccaactc catcactagg ggttcctgga gctccggtta cctacaaaat
      421 cagaaggaca gggaagggag cagtggttca cgcctgtaat cccagcaatt tgggaggcca
      481 aggtgggtag atcacctgag attaggagtt ggagaccagc ctggccaata tggtgaaacc
      541 ccgtctctac caaaaaaaca aaaattagct gagcctggtc atgcatgcct ggaatcccaa
      601 caactcggga ggctgaggca ggagaatcgc ttgaacccag gaggcggaga ttgcagtgag
      661 ccaagattgt gccactgcac tccagcttgg ttcccaatag accccgcagg ccctacaggt
      721 tgtcttccca acttgcccct tgctccatac cacccccctc caccccataa tattatagaa
      781 ggacacctag tcagacaaaa tgatgcaact taattttatt aggacaaggc tggtgggcac
      841 tggagtggca acttccaggg ccaggagagg cactggggag gggtcacagg gatgccaccc
      901 gtagatcgat ctacggtatc gatgcgggga ggcggcccaa agggagatcc gactcgtctg
      961 agggcgaagg cgaagacgcg gaagaggccg cagagccggc agcaggccgc gggaaggaag
     1021 gtccgctgga ttgagggccg aagggacgta gcagaaggac gtcccgcgca gaatccaggt
     1081 ggcaacacag gcgagcagcc aaggaaagga cgatgatttc cccgacaaca ccacggaatt
     1141 gtcagtgccc aacagccgag cccctgtcca gcagcgggca aggcaggcgg cgatgagttc
     1201 cgccgtggca atagggaggg ggaaagcgaa agtcccggaa aggagctgac aggtggtggc
     1261 aatgccccaa ccagtggggg ttgcgtcagc aaacacagtg cacaccacgc cacgttgcct
     1321 gacaacgggc cacaactcct cataaagaga cagcaaccag gatttataca aggaggagaa
     1381 aatgaaagcc atacgggaag caatagcatg atacaaaggc attaaagcag cgtatccaca
     1441 tagcgtaaaa ggagcaacat agttaagaat accagtcaat ctttcacaaa ttttgtaatc
     1501 cagaggttga ttatcgatcg gccgcatata agcttcgtac ggaagttact attccgaagt
     1561 tcctattctc tagaaagtat aggaacttcg tgtcgatggt gatgcttggc aattcgggtc
     1621 gatggtgaag cattggtctt cctattttga tgaagttcct attcttcaaa aggtatagga
     1681 acttcctcga gaccggtact agtgccacca tggtgagcaa gggcgaggag ctgttcaccg
     1741 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1801 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1861 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1921 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1981 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2041 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2101 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2161 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2221 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2281 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2341 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2401 ctctcggcat ggacgagctg tacaagtaag catgcatgaa ttcataactt cgtataatgt
     2461 atgctatacg aagttatttt ctttctttgc gcgtcctgga ttcgcggaaa cgcgtacatc
     2521 cagctgacga gtcccaaata ggacgaaacg cgcttttctt tttgttttta taacttcgta
     2581 taatgtatgc tatacgaagt tatcttaagg tcgacgaagt tcctatactt tctagagaat
     2641 aggaacttcg gaataggaac ttcgtgtcga tggtgatgct tggcaattcg ggtcgatggt
     2701 gaagcattgg tcttcctatt ttgatgaagt tcctatacct tttgaagaat aggaacttcg
     2761 ctagcggtac ctcacgacac ctgaaatgga agaaaaaaac tttgaaccac tgtctgaggc
     2821 ttgagaatga accaagatcc aaactcaaaa agggcaaatt ccaaggagaa ttacatcaag
     2881 tgccaagctg gcctaacttc agtctccacc cactcagtgt ggggaaactc catcgcataa
     2941 aacccctccc cccaacctaa agacgacgta ctccaaaagc tcgagaacta atcgaggtgc
     3001 ctggacggcg cccggtactc cgtggagtca catgaagcga cggctgagga cggaaaggcc
     3061 cttttccttt gtgtgggtga ctcacccgcc cgctctcccg agcgccgcgt cctccatttt
     3121 gagctccctg cagcagggcc gggaagcggc catctttccg ctcacgcaac tggtgccgac
     3181 cgggccagcc ttgccgccca gggcggggcg atacacggcg gcgcgaggcc aggcaccaga
     3241 gcaggccggc cagcttgaga ctacccccgt ccgattctcg gtggccgcgc tcgcaggccc
     3301 cgcctcgccg aacatgtgcg ctgggacgca cgggccccgt cgccgcccgc ggccccaaaa
     3361 accgaaatac cagtgtgcag atcttggccc gcatttacaa gactatcttg ccagaaaaaa
     3421 agcgtcgcag caggtcatca aaaattttaa atggctagag acttatcgaa agcagcgaga
     3481 caggcgcgaa ggtgccacca gattcgcacg cggcggcccc agcgcccagg ccaggcctca
     3541 actcaagcac gaggcgaagg ggctccttaa gcgcaaggcc tcgaactctc ccacccactt
     3601 ccaacccgaa gctcgggatc aagaatcacg tactgcagcc aggtggaagt aattcaaggc
     3661 acgcaagggc cataacccgt aaagaggcca ggcccgcggg aaccacacac ggcacttacc
     3721 tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca cggcgactac tgcacttata
     3781 tacggttctc ccccaccctc gggaaaaagg cggagccagt acacgacatc actttcccag
     3841 tttaccccgc gccaccttct ctaggcaccg gttcaattgc cgacccctcc ccccaacttc
     3901 tcggggactg tgggcgatgt gcgctctgcc cactgacggg caccggagcc aattcccact
     3961 cctttcaaga cctagaaggt ccattagctg caaagattcc tctctgttta aaactttatc
     4021 catctttgca aagcttacgc gtgcggccgc aggaacccct agtgatggag ttggccactc
     4081 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4141 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg attggatccc aatacgcgtc
     4201 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4261 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4321 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
     4381 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
     4441 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4501 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgacagtctc cgggagctgc
     4561 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacaaaaggg cctcgtgata
     4621 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     4681 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4741 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4801 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4861 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4921 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4981 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5041 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5101 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5161 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5221 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5281 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5341 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5401 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5461 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggttct
     5521 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5581 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5641 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5701 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5761 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5821 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5881 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5941 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6001 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6061 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6121 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6181 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6241 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6301 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6361 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6421 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6481 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     6541 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     6601 gagcg
//
