LOCUS       Exported                7166 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Intersectional viral expression of GCaMP6M in cells expressing Cre 
            AND NOT Flp.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a-Con/Foff 2.0-GCaMP6M
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7166)
  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 7166)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7166
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1426..1459
                     /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    1510..1543
                     /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(1636..1657)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(1870..1926)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     regulatory      1938..1947
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(1954..1987)
                     /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(2038..2071)
                     /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    2078..2111
                     /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     complement(2305..2326)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2566..2585)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(3151..3184)
                     /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    3293..3881
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3346..3366)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3764..3775)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3913..4389
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4112..4131)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4429..4569
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4429..4558
                     /label=AAV2 ITR
     rep_origin      4644..5099
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4731..4750)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4941..4962
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5116..5135)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5235..5257
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5295..5313)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5381..5485
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5486..6346
                     /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(5704..5723)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6517..7105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7006..7025
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gatccataac ttcgtatagg
     1441 atactttata cgaagttatg tagctggagc aatttgaaaa gattgttgta ttacctctag
     1501 ctgcgcttaa taacttcgta tagtatacct tatacgaagt tatgtcgacc ctcctgtact
     1561 caccttgctc accatgctcc ctccggtacc gcccttgtac agctcgtcca tgccgagagt
     1621 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     1681 tttcgaaagt ttggactgca cgctcaggta gtggttgtcg ggcagcagca cggggccgtc
     1741 gccgatgggg gtgttctgct ggtagtggta ggcgagctgc acgccgccgt cctcgatgtt
     1801 gtggcggatc ttgaagttcg ccttgatgcc gttcttctgc ttgtcggcct tgatatagac
     1861 gttctcgagt gagctcagcc gacctatagc tctgactgcg tgacctgtct tattccactt
     1921 acgacgtgat gagtccacca tggtggcact agtataactt cgtataaagt atcctatacg
     1981 aagttatttg ccttaaccca gaaattatca ctgttattct ttagataggt gcaaagaata
     2041 acttcgtata aggtatacta tacgaagtta tggtaccgaa gttcctattc tctagaaagt
     2101 ataggaactt cgcagaatgg tagctggatt gtagctgcta ttagcaatat gaaacctctt
     2161 agaagttcct attcttcaaa aggtatagga acttcggcgc gcctggagag tggccagccc
     2221 ttctcatgtt cagagaacat ggttaactgg ttaagtcatg tcgtcccaca gggcgaggag
     2281 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2341 ttcagcgtgt ccggcgaggg tgagggcgat gccacctacg gcaagctgac cctgaagttc
     2401 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2461 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2521 gccatgcccg aaggctacat ccaggagcgc accatcttct tcaaggacga cggcaactac
     2581 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2641 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacctgccg
     2701 gaccaactga ctgaagagca gatcgcagaa tttaaagagg ctttctccct atttgacaag
     2761 gacggggatg ggacaataac aaccaaggag ctggggacgg tgatgcggtc tctggggcag
     2821 aaccccacag aagcagagct gcaggacatg atcaatgaag tagatgccga cggtgacggc
     2881 acaatcgact tccctgagtt cctgacaatg atggcaagaa aagggagcta cagggacacg
     2941 gaagaagaaa ttagagaagc gttcggtgtg tttgataagg atggcaatgg ctacatcagt
     3001 gcagcagagc ttcgccacgt gatgacaaac cttggagaga agttaacaga tgaagaggtt
     3061 gatgaaatga tcagggaagc agacatcgat ggggatggtc aggtaaacta cgaagagttt
     3121 gtacaaatga tgacagcgaa gtgagctagc gaagttccta tactttctag agaataggaa
     3181 cttctaagca gacaatcttg taatacatca gcagctagta gattgctcca gctagaagtt
     3241 cctatacctt ttgaagaata ggaacttcga attcgatatc aagcttatcg ataatcaacc
     3301 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     3361 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     3421 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3481 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3541 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3601 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3661 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt
     3721 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3781 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3841 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct
     3901 cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa
     3961 gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct
     4021 gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa
     4081 gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg
     4141 gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag
     4201 cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt
     4261 tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg
     4321 atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc
     4381 cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag
     4441 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4501 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     4561 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4621 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4681 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4741 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4801 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     4861 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4921 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4981 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5041 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     5101 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     5161 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5221 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5281 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5341 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5401 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5461 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5521 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5581 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5641 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5701 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5761 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5821 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5881 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5941 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6001 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6061 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6121 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6181 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6241 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6301 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6361 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6421 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6481 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6541 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6601 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6661 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6721 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6781 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6841 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6901 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6961 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7021 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7081 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7141 cttttgctgg ccttttgctc acatgt
//
