LOCUS       sequence-271928-        7157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of sRGECO in cells expressing both 
            Cre AND Flp.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7157)
  AUTHORS   Fenno LE, Ramakrishnan C, Kim YS, Evans KE, Lo M, Vesuna S, Inoue M,
            Cheung KYM, Yuen E, Pichamoorthy N, Hong ASO, Deisseroth K
  TITLE     Comprehensive Dual- and Triple-Feature Intersectional Single-Vector 
            Delivery of Diverse Functional Payloads to Cells of Behaving 
            Mammals.
  JOURNAL   Neuron. 2020 Jun 20. pii: S0896-6273(20)30432-3. doi: 
            10.1016/j.neuron.2020.06.003.
  PUBMED    32574559
REFERENCE   2  (bases 1 to 7157)
  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: "Neuron. 
            2020 Jun 20. pii: S0896-6273(20)30432-3. doi: 
            10.1016/j.neuron.2020.06.003."
COMMENT     Sequence Label: pAAV-Ef1a-Con/Fon-sRGECO
FEATURES             Location/Qualifiers
     source          1..7157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1416..1449)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1500..1533)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     CDS             complement(1828..1884)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     regulatory      1896..1905
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1910..1943
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1994..2027
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    2034..2067
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     primer_bind     complement(2627..2646)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(3142..3175)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     misc_feature    3284..3872
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3337..3357)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3755..3766)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3904..4380
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4103..4122)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4420..4560
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4420..4549
                     /label=AAV2 ITR
     rep_origin      4635..5090
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4722..4741)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4932..4953
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5107..5126)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5226..5248
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5286..5304)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5372..5476
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5477..5545,5546..6337)
                     /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(5695..5714)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6508..7096
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6997..7016
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gatccataac ttcgtatagg atactttata
     1441 cgaagttatg tagctggagc aatttgaaaa gattgttgta ttacctctag ctgcgcttaa
     1501 taacttcgta tagtatacct tatacgaagt tatgtcgacc ctcctgtact cacctgtacc
     1561 tcccttgtac agctcgtcca tgccgccggt ggagtggcgg ccctcggcgc gttcgcactg
     1621 ttccacgatg gtgtagtcct cgttgtggga cacgatgtcc aacttgatgt ccacgatgta
     1681 ggcgccgggc agctgcacgg gcttcttggc cttgtaggtg gtcttgacct cggcggcgta
     1741 gtggccgccg tccttcagcc tcagcccctt cttgatctcg ctcttcaggg cgccgtcctc
     1801 ggggtacatc cgctcggaaa ccacgggtga gctcagccga cctatagctc tgactgcgtg
     1861 acctgcctta ttccacttac gacgtgatga gtccaccatg gtggctagca taacttcgta
     1921 taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt tattctttag
     1981 ataggtgcaa agaataactt cgtataaggt atactatacg aagttatggt accgaagttc
     2041 ctattcttca aatagtatag gaacttcaat tgttattggt gcaaagaatc acttccttaa
     2101 gtctttatac tgcccaggaa gttcctattc ttcaaaaggt ataggaactt cggcgcgccc
     2161 tacttcgctg tcatcatttg tacaaactct tcgtagttta cctgaccatc cccatcgatg
     2221 tctgctaccc tgatcatttc atcaacctcc tcatctgtta acttctctcc aaggtctgtc
     2281 atcacgtggc gaagctctgc tgcgccgatg tagccattgc cgtccttatc aaacacgcgg
     2341 aacgcttcgc gaatttcctc ttcactgtct gtgtcattca tttttcttgc catcatcgtc
     2401 aggaactcag ggaagtcgaa tgtgccgtca ccgtcggcat ctacttcatt gatcatgtcc
     2461 tgcagctctg cttctgtggg gttctgcccc agcgatcgga acaccgtccc aagctccttg
     2521 gttgttatcg tcccatcccc gtccttgtca aatagggaga aagcctcttt aaattctgcg
     2581 atctgctctt cagtcaggtc gtcacgcgta gcctcccagc ccatggtctt cttctgcatt
     2641 acggggccgt cgggggggaa gttggtgccg cgcagcttca ccttgtagat gaatacgccg
     2701 tcctgcaggg aggagtcctg gttcacgtga ataatgccgc cgtcctcgaa gttcatcacg
     2761 cggtcccacc tgaagccctc ggggaaggac agcttgaagt agtcggggat gtcggctggg
     2821 tgcttaatgt aggccttgga gccgtacatg aactgagggg acaggatgtc ccaggcgaag
     2881 ggcagggggc cacccttggt caccttcagc ttagcggtct gaaaggcctc gtaggggcgg
     2941 ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc catgtgcacc
     3001 ttgaagcgca tgaactcctt gatgatggcc atgttatcct cctcgccctt gctcaccaga
     3061 ctcccgcctg tgggacgaca tgacttaacc agttaaccat gttctctgaa catgagaagg
     3121 gctggccact ctccatctag agaagttcct atactatttg aagaatagga acttcttgcc
     3181 ttaacccaga aattatcact gttattcttt agaatagtgc aaagagaagt tcctatacct
     3241 tttgaagaat aggaacttcg aattcgatat caagcttatc gataatcaac ctctggatta
     3301 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     3361 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     3421 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     3481 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     3541 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     3601 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     3661 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg
     3721 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     3781 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     3841 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc
     3901 tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
     3961 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
     4021 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
     4081 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
     4141 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
     4201 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga
     4261 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca
     4321 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt
     4381 ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt
     4441 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     4501 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     4561 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     4621 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4681 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     4741 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4801 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     4861 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4921 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     4981 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     5041 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     5101 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     5161 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5221 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     5281 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     5341 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5401 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5461 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5521 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5581 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5641 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5701 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5761 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5821 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5881 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5941 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6001 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6061 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6121 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6181 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6241 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6301 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6361 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6421 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6481 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6541 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6601 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     6661 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6721 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6781 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6841 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6901 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6961 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7021 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7081 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7141 gccttttgct cacatgt
//
