LOCUS       Exported                6715 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  For co-expression of GCaMP6s with mKate2.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-hSynapsin1-GCaMP6s-P2A-mKate2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6715)
  AUTHORS   Broussard GJ, Liang Y, Fridman M, Unger EK, Meng G, Xiao X, Ji N, 
            Petreanu L, Tian L
  TITLE     In vivo measurement of afferent activity with axon-specific calcium 
            imaging.
  JOURNAL   Nat Neurosci. 2018 Sep;21(9):1272-1280. doi: 
            10.1038/s41593-018-0211-4. Epub 2018 Aug 20.
  PUBMED    30127424
REFERENCE   2  (bases 1 to 6715)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6715
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   26..155
                     /label=AAV2 ITR
     promoter        237..684
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      715..724
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             721..2070
                     /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"
     regulatory      817..826
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     1107..1128
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1234..1255)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1495..1514)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2080..2136
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2137..2832
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     misc_feature    2853..3441
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2906..2926)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3324..3335)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3444..3460)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3445..3461)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3490..3611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3501..3520)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3555..3574
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3773..3902
                     /label=AAV2 ITR
     primer_bind     complement(3921..3938)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3921..3937)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3930..3952)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4079..4534
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4166..4185)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4376..4397
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4560..4664
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4665..5525
                     /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(4883..4902)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5696..6284
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6185..6204
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6438..6455
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6572..6593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6608..6638
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6646..6662
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     6651..6673
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6670..6686
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6670..6686
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cgcagaattt aaagaggtaa ttaggctgcg cgctcgctcg ctcactgagg ccgcccgggc
       61 aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
      121 agagggagtg gccaactcca tcactagggg ttccttgtag ttaatgatta acccgccatg
      181 ctacttatct acgtagccat gctctaggaa gatctctgca gagggccctg cgtatgagtg
      241 caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac gaccgacccc
      301 gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg
      361 gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca
      421 gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg
      481 acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc
      541 gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac
      601 catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggagga
      661 gtcgtgtcgt gcctgagagc gcagtcgaat tcaagctgct agctagtacc ggtcgccacc
      721 atgggttctc atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg
      781 ggtcgggatc tgtacgacga tgacgataag gatctcgcca ccatggtcga ctcatcacgt
      841 cgtaagtgga ataagacagg tcacgcagtc agagctatag gtcggctgag ctcactcgag
      901 aacgtctata tcaaggccga caagcagaag aacggcatca aggcgaactt ccacatccgc
      961 cacaacatcg aggacggcgg cgtgcagctc gcctaccact accagcagaa cacccccatc
     1021 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcgtgcagtc caaactttcg
     1081 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1141 atcactctcg gcatggacga gctgtacaag ggcggtaccg gagggagcat ggtgagcaag
     1201 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1261 ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg caagctgacc
     1321 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1381 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1441 ttcaagtccg ccatgcccga aggctacatc caggagcgca ccatcttctt caaggacgac
     1501 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1561 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1621 aacctgccgg accaactgac tgaagagcag atcgcagaat ttaaagaggc tttctcccta
     1681 tttgacaagg acggggatgg gacaataaca accaaggagc tggggacggt gatgcggtct
     1741 ctggggcaga accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac
     1801 ggtgacggca caatcgactt ccctgagttc ctgacaatga tggcaagaaa aatgaaatac
     1861 agggacacgg aagaagaaat tagagaagcg ttcggtgtgt ttgataagga tggcaatggc
     1921 tacatcagtg cagcagagct tcgccacgtg atgacaaacc ttggagagaa gttaacagat
     1981 gaagaggttg atgaaatgat cagggaagca gacatcgatg gggatggtca ggtaaactac
     2041 gaagagtttg tacaaatgat gacagcgaag ggatccggag ccacgaactt ctctctgtta
     2101 aagcaagcag gagacgtgga agaaaacccc ggtcctgtga gcgagctgat taaggagaac
     2161 atgcacatga agctgtacat ggagggcacc gtgaacaacc accacttcaa gtgcacatcc
     2221 gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggc ggtcgagggc
     2281 ggccctctcc ccttcgcctt cgacatcctg gctaccagct tcatgtacgg cagcaaaacc
     2341 ttcatcaacc acacccaggg catccccgac ttctttaagc agtccttccc cgagggcttc
     2401 acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc
     2461 agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa cttcccatcc
     2521 aacggccctg tgatgcagaa gaaaacactc ggctgggagg cctccaccga gaccctgtac
     2581 cccgctgacg gcggcctgga aggcagagcc gacatggccc tgaagctcgt gggcgggggc
     2641 cacctgatct gcaacttgaa gaccacatac agatccaaga aacccgctaa gaacctcaag
     2701 atgcccggcg tctactatgt ggacagaaga ctggaaagaa tcaaggaggc cgacaaagag
     2761 acctacgtcg agcagcacga ggtggctgtg gccagatact gcgacctccc tagcaaactg
     2821 gggcacagat aagcttacta gagcttatcg ataatcaacc tctggattac aaaatttgtg
     2881 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     2941 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     3001 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     3061 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     3121 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     3181 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     3241 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     3301 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     3361 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     3421 ccctttgggc cgcctccccg catcgatacc gtcgacctcg acccgggcgg ccgcttcgag
     3481 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     3541 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     3601 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt
     3661 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatcttc
     3721 ctagagcatg gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc
     3781 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     3841 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     3901 agccttaatt aacctaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     3961 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4021 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     4081 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4141 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4201 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4261 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4321 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4381 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4441 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4501 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     4561 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4621 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4681 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4741 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4801 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4861 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4921 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4981 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5041 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5101 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5161 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5221 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5281 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5341 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5401 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5461 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5521 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5581 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5641 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5701 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5761 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5821 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5881 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5941 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6001 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6061 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6121 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6181 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6241 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6301 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6361 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6421 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     6481 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     6541 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     6601 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     6661 aatttcacac aggaaacagc tatgaccatg attacgccag atttaattaa ggcct
//
