LOCUS       Exported                5917 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV expression of Cre recombinase-activated ultrasensitive protein 
            calcium sensor from Synapsin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV.Syn.Flex.GCaMP6s.WPRE.SV40
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5917)
  AUTHORS   Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, 
            Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, 
            Kim DS
  TITLE     Ultrasensitive fluorescent proteins for imaging neuronal activity.
  JOURNAL   Nature. 2013 Jul 18;499(7458):295-300. doi: 10.1038/nature12354.
  PUBMED    23868258
REFERENCE   2  (bases 1 to 5917)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5917
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   65..194
                     /label=AAV2 ITR
     promoter        276..723
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(753..786)
                     /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(849..882)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(893..2245)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     1452..1471
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1711..1732
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1838..1859)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2140..2149
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      2242..2251
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    2262..2295
                     /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    2366..2399
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    2441..3029
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2494..2514)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2912..2923)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3032..3048)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3033..3049)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3078..3199
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3089..3108)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3143..3162
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3534..3551)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3705..4293)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3785..3804)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4464..5324)
                     /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     5087..5106
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5325..5429)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5455..5910)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5592..5613)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5804..5823
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
       61 ccagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac
      121 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
      181 cactaggggt tccttgtagt taatgattaa cccgccatgc tacttatcta cgtagccatg
      241 ctctaggaag atctctgcag agggccctgc gtatgagtgc aagtgggttt taggaccagg
      301 atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga caagcaccca
      361 acccccattc cccaaattgc gcatccccta tcagagaggg ggaggggaaa caggatgcgg
      421 cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc cccgcctggc
      481 ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc cggtcccccg
      541 caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc agccggaccg
      601 caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg cggcgccggc
      661 gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg cctgagagcg
      721 cagtcgaatt caagctgcta gcaaggatcc ccataacttc gtataaagta tcctatacga
      781 agttatatca aaataggaag accaatgctt caccatcgac ccgaattgcc aagcatcacc
      841 atcgacccat aacttcgtat aatgtatgct atacgaagtt atactagagc tttcacttcg
      901 ctgtcatcat ttgtacaaac tcttcgtagt ttacctgacc atccccatcg atgtctgctt
      961 ccctgatcat ttcatcaacc tcttcatctg ttaacttctc tccaaggttt gtcatcacgt
     1021 ggcgaagctc tgctgcactg atgtagccat tgccatcctt atcaaacaca ccgaacgctt
     1081 ctctaatttc ttcttccgtg tccctgtatt tcatttttct tgccatcatt gtcaggaact
     1141 cagggaagtc gattgtgccg tcaccgtcgg catctacttc attgatcatg tcctgcagct
     1201 ctgcttctgt ggggttctgc cccagagacc gcatcaccgt ccccagctcc ttggttgtta
     1261 ttgtcccatc cccgtccttg tcaaataggg agaaagcctc tttaaattct gcgatctgct
     1321 cttcagtcag ttggtccggc aggttgtact ccagcttgtg ccccaggatg ttgccgtcct
     1381 ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca
     1441 cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggatgt
     1501 agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc
     1561 tgaagcactg cacgccgtag gtcagggtgg tcacgagggt gggccagggc acgggcagct
     1621 tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcaccct
     1681 cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca
     1741 ccaccccggt gaacagctcc tcgcccttgc tcaccatgct ccctccggta ccgcccttgt
     1801 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt
     1861 gatcgcgctt ctcgttgggg tctttcgaaa gtttggactg cacgctcagg tagtggttgt
     1921 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg taggcgagct
     1981 gcacgccgcc gtcctcgatg ttgtggcgga tgtggaagtt cgccttgatg ccgttcttct
     2041 gcttgtcggc cttgatatag acgttctcga gtgagctcag ccgacctata gctctgactg
     2101 cgtgacctgt cttattccac ttacgacgtg atgagtcgac catggtggcg agatccttat
     2161 cgtcatcgtc gtacagatcc cgacccattt gctgtccacc agtcatgcta gccataccat
     2221 gatgatgatg atgatgagaa cccatggtgg cggatcctag tataacttcg tataggatac
     2281 tttatacgaa gttatcattg ggattcttcc tattttgatc caagcatcac catcgaccct
     2341 ctagtccaga tctcaccatc gacccataac ttcgtatagc atacattata cgaagttatg
     2401 tccctcgaag aggttcgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa
     2461 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     2521 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     2581 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     2641 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     2701 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     2761 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     2821 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     2881 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     2941 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     3001 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgac ccgggcggcc
     3061 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     3121 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     3181 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     3241 ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa
     3301 ggatcttcct agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca
     3361 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     3421 ccgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcattaatga
     3481 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3541 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3601 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3661 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3721 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3781 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3841 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3901 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3961 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4021 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4081 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4141 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4201 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4261 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4321 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4381 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4441 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4501 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4561 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4621 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4681 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4741 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4801 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4861 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4921 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4981 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5041 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5101 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5161 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5221 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5281 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5341 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5401 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     5461 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     5521 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     5581 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     5641 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     5701 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     5761 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     5821 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     5881 ccgcgcttaa tgcgccgcta cagggcgcgt cccattc
//
