LOCUS       Exported                6778 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Fluorescent reporter for calcium imaging and photoactivation..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV2-GCaMP5g-P2A-PAmCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6778)
  AUTHORS   Lee D, Kume M, Holy TE
  TITLE     Sensory coding mechanisms revealed by optical tagging of 
            physiologically defined neuronal types.
  JOURNAL   Science. 2019 Dec 13;366(6471):1384-1389. doi: 
            10.1126/science.aax8055.
  PUBMED    31831669
REFERENCE   2  (bases 1 to 6778)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6778
                     /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
     enhancer        235..538
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        540..817
                     /label=chicken beta-actin promoter
     primer_bind     1074..1093
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1148..1157
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1163..1180
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1184..1216
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1220..1243
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      1250..1259
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1271..1327
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     1540..1561
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1667..1688)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1928..1947)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2063..2503
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAEL
                     RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             2519..2575
                     /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"
     regulatory      2582..2591
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2588..3298
                     /codon_start=1
                     /product="photoactivatable monomeric derivative of DsRed 
                     fluorescent protein"
                     /label=PAmCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHVFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFTWDILSPQFMYGSNAYVKHPADIPDYFKLSFPEGFKWERVMKF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEALSERMYPEDGALK
                     GEVKPRVKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNRKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2733..2751)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3001..3020
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3324..3445
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3335..3354)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3389..3408
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3531..3548)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3537..3744
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3774..3914
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3774..3903
                     /label=AAV2 ITR
     rep_origin      4010..4465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4097..4116)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4307..4328
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4532..4551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4651..4673
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4711..4729)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4797..4901
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4902..5762
                     /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(5120..5139)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5933..6521
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6422..6441
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6675..6692
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtagatc tgaattcggt accctagtta
      181 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      241 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      301 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      361 ggactattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      421 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      481 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      541 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
      601 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
      661 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc
      721 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
      781 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga cgctgccttc
      841 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
      901 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg
      961 tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagc
     1021 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt
     1081 gctggttatt gtgctgtctc atcattttgg caaagaattc ctcgaagatc cgaaggggtt
     1141 caagcttgcc accatgggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
     1201 tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatctcg ccaccatggt
     1261 cgactcatca cgtcgtaagt ggaataagac aggtcacgca gtcagagcta taggtcggct
     1321 gagctcactc gagaacgtct atatcaaggc cgacaagcag aagaacggca tcaaggcgaa
     1381 cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc actaccagca
     1441 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcgtgca
     1501 gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1561 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta ccggagggag
     1621 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1681 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggtgagggcg atgccaccta
     1741 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1801 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1861 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac atccaggagc gcaccatctt
     1921 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1981 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2041 caagctggag tacaacctgc cggaccaact gactgaagag cagatcgcag aatttaaaga
     2101 ggctttctcc ctatttgaca aggacgggga tgggacaata acaaccaagg agctggggac
     2161 ggtgatgcgg tctctggggc agaaccccac agaagcagag ctgcaggaca tgatcaatga
     2221 agtagatgcc gacggtgacg gcacaatcga cttccctgag ttcctgacaa tgatggcaag
     2281 aaaaatgaaa tacacagaca gtgaagaaga aattagagaa gcgttccgtg tgtttgataa
     2341 ggatggcaat ggctacatca gtgcagcaga gcttcgccac gtgatgacaa accttggaga
     2401 gaagttaaca gatgaagagg ttgatgaaat gatcagggaa gcagacatcg atggggatgg
     2461 tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg aaggaattcg gaagcggagc
     2521 tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg gacctggatc
     2581 cgccaccatg gtgagcaagg gcgaggagga taacatggcc atcattaagg agttcatgcg
     2641 cttcaaggtg cacatggagg ggtccgtgaa cggccacgtg ttcgagatcg agggcgaggg
     2701 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
     2761 cctgcccttc acctgggaca tcctgtcccc tcaattcatg tacggctcca atgcctacgt
     2821 gaagcacccc gccgacatcc ccgactactt taagctgtcc ttccccgagg gcttcaagtg
     2881 ggagcgcgtg atgaaattcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     2941 gcaggacggt gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
     3001 ccccgtaatg cagaagaaga ccatgggctg ggaggccctc tccgagcgga tgtaccccga
     3061 ggacggcgcc ctgaagggcg aggtcaagcc gagagtgaag ctgaaggacg gcggccacta
     3121 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta
     3181 caacgtcaac cgcaagttgg acatcacctc acacaacgag gactacacca tcgtggaaca
     3241 gtacgaacgt gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaagc
     3301 ggccgcgcgg atcccagaca tgataagata cattgatgag tttggacaaa ccacaactag
     3361 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     3421 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     3481 tcagggggag gtgtgggagg ttttttagtc gactagagct cgctgatcag cctcgactgt
     3541 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3601 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3661 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3721 agacaatagc aggcatgctg gggagagatc tcacgtgcgg accgagcggc cgcaggaacc
     3781 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     3841 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     3901 cagctgcctg caggggcgcc tgatgctgca gccagctggc gtaatagcga agaggcccgc
     3961 accgatcgcc cttcccaaca gttgcgtagc ctgaatggcg aatggcgcga cgcgccctgt
     4021 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4081 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4141 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4201 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     4261 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4321 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     4381 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4441 aacaaaatat taacgtttac aatttcctga tgcggtattt tctccttacg catctgtgcg
     4501 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4561 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4621 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4681 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     4741 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     4801 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4861 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4921 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4981 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5041 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5101 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5161 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5221 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5281 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5341 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5401 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5461 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5521 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5581 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5641 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5701 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5761 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5821 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5881 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5941 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6001 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6061 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6121 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6181 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6241 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6301 aactgagata cctacagcgt gagcattgag aaagcgccac gcttcccgaa gggagaaagg
     6361 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6421 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6481 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6541 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6601 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6661 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     6721 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg ggctgcaggc tcacatgt
//
