LOCUS       Exported                7159 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Intersectional viral expression of sRGECO in cells expressing Cre 
            AND NOT Flp.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a-Con/Foff 2.0-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"
     rep_origin      166..621
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(253..272)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     463..484
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(638..657)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     757..779
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(817..835)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        903..1007
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1008..1868
                     /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(1226..1245)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2039..2627
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2528..2547
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2689..2818
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2919..4097
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3150..4088
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4045..4065
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    4104..4137
                     /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    4188..4221
                     /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."
     CDS             complement(4516..4572)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     regulatory      4584..4593
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(4600..4633)
                     /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(4684..4717)
                     /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."
     protein_bind    4724..4757
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     5339..5358
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5832..5865)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    5974..6562
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6027..6047)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6445..6456)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6594..7070
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(6793..6812)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   7110..91
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7110..80
                     /label=AAV2 ITR
ORIGIN
        1 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
       61 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
      121 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
      181 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      241 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      301 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
      361 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
      421 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
      481 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
      541 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
      601 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
      661 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
      721 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
      781 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
      841 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
      901 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
      961 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1021 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1081 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1141 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1201 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     1261 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     1321 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     1381 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     1441 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     1501 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     1561 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     1621 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     1681 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     1741 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     1801 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     1861 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     1921 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     1981 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2041 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2101 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2161 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     2221 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2281 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2341 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2401 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2461 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2521 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2581 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2641 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc
     2701 tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc
     2761 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc
     2821 ggccgcacgc gtaagctttg caaagatgga taaagtttta aacagagagg aatctttgca
     2881 gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg
     2941 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
     3001 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
     3061 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     3121 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
     3181 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg
     3241 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
     3301 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
     3361 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
     3421 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     3481 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     3541 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     3601 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     3661 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     3721 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     3781 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     3841 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     3901 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     3961 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     4021 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     4081 catttcaggt gtcgtgagga tccataactt cgtataggat actttatacg aagttatgta
     4141 gctggagcaa tttgaaaaga ttgttgtatt acctctagct gcgcttaata acttcgtata
     4201 gtatacctta tacgaagtta tgtcgaccct cctgtactca cctgtacctc ccttgtacag
     4261 ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt tcgcactgtt ccacgatggt
     4321 gtagtcctcg ttgtgggaca cgatgtccaa cttgatgtcc acgatgtagg cgccgggcag
     4381 ctgcacgggc ttcttggcct tgtaggtggt cttgacctcg gcggcgtagt ggccgccgtc
     4441 cttcagcctc agccccttct tgatctcgct cttcagggcg ccgtcctcgg ggtacatccg
     4501 ctcggaaacc acgggtgagc tcagccgacc tatagctctg actgcgtgac ctgccttatt
     4561 ccacttacga cgtgatgagt ccaccatggt ggcactagta taacttcgta taaagtatcc
     4621 tatacgaagt tatttgcctt aacccagaaa ttatcactgt tattctttag ataggtgcaa
     4681 agaataactt cgtataaggt atactatacg aagttatggt accgaagttc ctattctcta
     4741 gaaagtatag gaacttcgca gaatggtagc tggattgtag ctgctattag caatatgaaa
     4801 cctcttagaa gttcctattc ttcaaaaggt ataggaactt cggcgcgcct ggagagtggc
     4861 cagcccttct catgttcaga gaacatggtt aactggttaa gtcatgtcgt cccacaggcg
     4921 ggagtctggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
     4981 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     5041 agggccgccc ctacgaggcc tttcagaccg ctaagctgaa ggtgaccaag ggtggccccc
     5101 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacatta
     5161 agcacccagc cgacatcccc gactacttca agctgtcctt ccccgagggc ttcaggtggg
     5221 accgcgtgat gaacttcgag gacggcggca ttattcacgt gaaccaggac tcctccctgc
     5281 aggacggcgt attcatctac aaggtgaagc tgcgcggcac caacttcccc cccgacggcc
     5341 ccgtaatgca gaagaagacc atgggctggg aggctacgcg tgacgacctg actgaagagc
     5401 agatcgcaga atttaaagag gctttctccc tatttgacaa ggacggggat gggacgataa
     5461 caaccaagga gcttgggacg gtgttccgat cgctggggca gaaccccaca gaagcagagc
     5521 tgcaggacat gatcaatgaa gtagatgccg acggtgacgg cacattcgac ttccctgagt
     5581 tcctgacgat gatggcaaga aaaatgaatg acacagacag tgaagaggaa attcgcgaag
     5641 cgttccgcgt gtttgataag gacggcaatg gctacatcgg cgcagcagag cttcgccacg
     5701 tgatgacaga ccttggagag aagttaacag atgaggaggt tgatgaaatg atcagggtag
     5761 cagacatcga tggggatggt caggtaaact acgaagagtt tgtacaaatg atgacagcga
     5821 agtaggctag cgaagttcct atactttcta gagaatagga acttctaagc agacaatctt
     5881 gtaatacatc agcagctagt agattgctcc agctagaagt tcctatacct tttgaagaat
     5941 aggaacttcg aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt
     6001 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     6061 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     6121 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     6181 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     6241 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     6301 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     6361 tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc
     6421 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     6481 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     6541 tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc tacgggtggc
     6601 atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca
     6661 ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata
     6721 atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta
     6781 gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg
     6841 caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat
     6901 tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca
     6961 ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc
     7021 ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt
     7081 aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc
     7141 ctctctgcgc gctcgctcg
//
