LOCUS       Exported                6694 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 6694)
  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 6694)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6694
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        212..659
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      690..699
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             696..2045
                     /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      792..801
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     1082..1103
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1209..1230)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1470..1489)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2055..2111
                     /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             2112..2807
                     /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    2828..3416
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2881..2901)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3299..3310)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3419..3435)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3420..3436)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3465..3586
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3476..3495)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3530..3549
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3851..3870)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3852..3870)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3877..3894)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3877..3893)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3886..3908)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4034..4489
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4121..4140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4331..4352
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4515..4619
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4620..5480
                     /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(4838..4857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5651..6239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6140..6159
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6393..6410
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6526..6547
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6562..6592
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagatct ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga
      241 ggcggggtgg gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc
      301 ccattcccca aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag
      361 gcgcgtgcgc actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg
      421 cgcgccaccg ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa
      481 ctccccttcc cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc
      541 acgcgaggcg cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact
      601 cagcgctgcc tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcgcagt
      661 cgaattcaag ctgctagcta gtaccggtcg ccaccatggg ttctcatcat catcatcatc
      721 atggtatggc tagcatgact ggtggacagc aaatgggtcg ggatctgtac gacgatgacg
      781 ataaggatct cgccaccatg gtcgactcat cacgtcgtaa gtggaataag acaggtcacg
      841 cagtcagagc tataggtcgg ctgagctcac tcgagaacgt ctatatcaag gccgacaagc
      901 agaagaacgg catcaaggcg aacttccaca tccgccacaa catcgaggac ggcggcgtgc
      961 agctcgccta ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     1021 acaaccacta cctgagcgtg cagtccaaac tttcgaaaga ccccaacgag aagcgcgatc
     1081 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     1141 acaagggcgg taccggaggg agcatggtga gcaagggcga ggagctgttc accggggtgg
     1201 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1261 agggtgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1321 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1381 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1441 acatccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1501 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1561 aggacggcaa catcctgggg cacaagctgg agtacaacct gccggaccaa ctgactgaag
     1621 agcagatcgc agaatttaaa gaggctttct ccctatttga caaggacggg gatgggacaa
     1681 taacaaccaa ggagctgggg acggtgatgc ggtctctggg gcagaacccc acagaagcag
     1741 agctgcagga catgatcaat gaagtagatg ccgacggtga cggcacaatc gacttccctg
     1801 agttcctgac aatgatggca agaaaaatga aatacaggga cacggaagaa gaaattagag
     1861 aagcgttcgg tgtgtttgat aaggatggca atggctacat cagtgcagca gagcttcgcc
     1921 acgtgatgac aaaccttgga gagaagttaa cagatgaaga ggttgatgaa atgatcaggg
     1981 aagcagacat cgatggggat ggtcaggtaa actacgaaga gtttgtacaa atgatgacag
     2041 cgaagggatc cggagccacg aacttctctc tgttaaagca agcaggagac gtggaagaaa
     2101 accccggtcc tgtgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg
     2161 gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg
     2221 gcacccagac catgagaatc aaggcggtcg agggcggccc tctccccttc gccttcgaca
     2281 tcctggctac cagcttcatg tacggcagca aaaccttcat caaccacacc cagggcatcc
     2341 ccgacttctt taagcagtcc ttccccgagg gcttcacatg ggagagagtc accacatacg
     2401 aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct
     2461 acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg cagaagaaaa
     2521 cactcggctg ggaggcctcc accgagaccc tgtaccccgc tgacggcggc ctggaaggca
     2581 gagccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac ttgaagacca
     2641 catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac tatgtggaca
     2701 gaagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag cacgaggtgg
     2761 ctgtggccag atactgcgac ctccctagca aactggggca cagataagct tactagagct
     2821 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     2881 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     2941 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3001 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3061 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcactttccc
     3121 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3181 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     3241 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3301 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3361 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     3421 ataccgtcga cctcgacccg ggcggccgct tcgagcagac atgataagat acattgatga
     3481 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     3541 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     3601 cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa
     3661 cctctacaaa tgtggtaaaa tcgataagga tcttcctaga gcatggctac gtagataagt
     3721 agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct
     3781 ctctgcgcgc tcgctcgctc actgaggccg cccgggcttt gcccgggcgg cctcagtgag
     3841 cggtatcacg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
     3901 tgactgggaa aaccctggcg tggcctwrcc cgggcggcct cagtgagcga gcgagcgcgc
     3961 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     4021 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4081 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4141 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4201 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4261 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4321 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4381 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4441 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     4501 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4561 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4621 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4681 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4741 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4801 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4861 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4921 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4981 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5041 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5101 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5161 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5221 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5281 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5341 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5401 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5461 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5521 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5581 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5641 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5701 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5761 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5821 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5881 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5941 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6001 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6061 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6121 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6181 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6241 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6301 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     6361 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     6421 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg
     6481 cgctcgctcg ctcgactggg acgcgggcag tgagcgcaac gcaattaatg tgagttagct
     6541 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     6601 tgtgagcggg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
     6661 gcgatcggtg cgggcctctt cgctattacg ccag
//
