LOCUS       Exported                7159 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Intersectional viral expression of sRGECO in cells expressing Flp 
            AND NOT Cre.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a-Coff/Fon-sRGECO
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7159)
  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 7159)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7159
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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    1416..1449
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    1500..1533
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GTATACCT). loxN sites are compatible with each 
                     other, but incompatible with loxP or lox2272 sites."
     regulatory      1540..1549
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1561..1617
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     protein_bind    complement(1912..1945)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(1996..2029)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GTATACCT). loxN sites are compatible with each 
                     other, but incompatible with loxP or lox2272 sites."
     primer_bind     complement(2629..2648)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3286..3874
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3339..3359)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3757..3768)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3906..4382
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4105..4124)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4422..4562
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4422..4551
                     /label=AAV2 ITR
     rep_origin      4637..5092
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4724..4743)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4934..4955
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5109..5128)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5228..5250
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5288..5306)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5374..5478
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5479..6339
                     /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(5697..5716)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6510..7098
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6999..7018
                     /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 tatgtcgacg ccaccatggt ggactcatca
     1561 cgtcgtaagt ggaataaggc aggtcacgca gtcagagcta taggtcggct gagctcaccc
     1621 gtggtttccg agcggatgta ccccgaggac ggcgccctga agagcgagat caagaagggg
     1681 ctgaggctga aggacggcgg ccactacgcc gccgaggtca agaccaccta caaggccaag
     1741 aagcccgtgc agctgcccgg cgcctacatc gtggacatca agttggacat cgtgtcccac
     1801 aacgaggact acaccatcgt ggaacagtgc gaacgcgccg agggccgcca ctccaccggc
     1861 ggcatggacg agctgtacaa gggaggtaca ggtgagtaca ggagggctag cataacttcg
     1921 tataaagtat cctatacgaa gttatttgcc ttaacccaga aattatcact gttattcttt
     1981 agataggtgc aaagaataac ttcgtataag gtatactata cgaagttatg gtaccgaagt
     2041 tcctattctt caaatagtat aggaacttca attgttattg gtgcaaagaa tcacttcctt
     2101 aagtctttat actgcccagg aagttcctat tcttcaaaag gtataggaac ttcggcgcgc
     2161 cctacttcgc tgtcatcatt tgtacaaact cttcgtagtt tacctgacca tccccatcga
     2221 tgtctgctac cctgatcatt tcatcaacct cctcatctgt taacttctct ccaaggtctg
     2281 tcatcacgtg gcgaagctct gctgcgccga tgtagccatt gccgtcctta tcaaacacgc
     2341 ggaacgcttc gcgaatttcc tcttcactgt ctgtgtcatt catttttctt gccatcatcg
     2401 tcaggaactc agggaagtcg aatgtgccgt caccgtcggc atctacttca ttgatcatgt
     2461 cctgcagctc tgcttctgtg gggttctgcc ccagcgatcg gaacaccgtc ccaagctcct
     2521 tggttgttat cgtcccatcc ccgtccttgt caaataggga gaaagcctct ttaaattctg
     2581 cgatctgctc ttcagtcagg tcgtcacgcg tagcctccca gcccatggtc ttcttctgca
     2641 ttacggggcc gtcggggggg aagttggtgc cgcgcagctt caccttgtag atgaatacgc
     2701 cgtcctgcag ggaggagtcc tggttcacgt gaataatgcc gccgtcctcg aagttcatca
     2761 cgcggtccca cctgaagccc tcggggaagg acagcttgaa gtagtcgggg atgtcggctg
     2821 ggtgcttaat gtaggccttg gagccgtaca tgaactgagg ggacaggatg tcccaggcga
     2881 agggcagggg gccacccttg gtcaccttca gcttagcggt ctgaaaggcc tcgtaggggc
     2941 ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc tccatgtgca
     3001 ccttgaagcg catgaactcc ttgatgatgg ccatgttatc ctcctcgccc ttgctcacca
     3061 gactcccgcc tgtgggacga catgacttaa ccagttaacc atgttctctg aacatgagaa
     3121 gggctggcca ctctccatct agagaagttc ctatactatt tgaagaatag gaacttcttg
     3181 ccttaaccca gaaattatca ctgttattct ttagaatagt gcaaagagaa gttcctatac
     3241 cttttgaaga ataggaactt cgaattcgat atcaagctta tcgataatca acctctggat
     3301 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3361 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3421 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     3481 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3541 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3601 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3661 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc
     3721 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3781 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3841 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga
     3901 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
     3961 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg
     4021 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga
     4081 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat
     4141 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg
     4201 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
     4261 gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc
     4321 caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc
     4381 ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     4441 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     4501 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     4561 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4621 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4681 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     4741 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4801 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4861 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4921 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     4981 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     5041 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     5101 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5161 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5221 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5281 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     5341 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5401 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5461 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5521 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5581 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5641 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5701 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5761 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5821 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5881 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5941 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6001 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6061 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6121 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6181 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6241 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6301 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6361 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6421 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6481 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6541 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6601 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6661 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6721 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6781 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6841 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6901 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6961 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7021 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7081 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7141 tggccttttg ctcacatgt
//
