LOCUS       Exported                7241 bp ds-DNA     circular SYN 14-FEB-2022
DEFINITION  Cre-activated AAV expression of humanized ChR2 with H134R mutation 
            fused to mCherry for optogenetic activation.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7241)
  TITLE     Cre-activated AAV expression plasmids
REFERENCE   2  (bases 1 to 7241)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 14, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7241
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   121..261
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   121..250
                     /label=AAV2 ITR
     rep_origin      336..791
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(423..442)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     633..654
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(808..827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     927..949
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(987..1005)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1073..1177
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1178..2038
                     /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(1396..1415)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2209..2797
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2698..2717
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2859..2988
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        3089..4267
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3320..4258
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4215..4235
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    4298..4331
                     /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    4382..4415
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(4427..5134)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(4705..4724)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4974..4992
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(5147..6073)
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      6070..6079
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(6084..6117)
                     /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(6168..6201)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    6226..6814
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6279..6299)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6697..6708)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6846..81
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(7045..7064)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
ORIGIN
        1 taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc
       61 actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc
      121 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
      181 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
      241 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
      301 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
      361 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
      421 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      481 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      541 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
      601 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
      661 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
      721 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
      781 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
      841 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
      901 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
      961 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     1021 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     1081 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1141 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     1201 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     1261 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     1321 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     1381 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     1441 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     1501 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     1561 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     1621 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     1681 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     1741 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     1801 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     1861 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     1921 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     1981 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     2041 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     2101 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     2161 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2221 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2281 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2341 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2401 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2461 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2521 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     2581 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     2641 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     2701 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     2761 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     2821 acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg
     2881 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     2941 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcacgc
     3001 gtaagctttg caaagatgga taaagtttta aacagagagg aatctttgca gctaatggac
     3061 cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac
     3121 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
     3181 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     3241 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     3301 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac
     3361 gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga
     3421 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc
     3481 ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg
     3541 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg
     3601 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct
     3661 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg
     3721 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc
     3781 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg
     3841 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg
     3901 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc
     3961 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga
     4021 gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt
     4081 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga
     4141 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg
     4201 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt
     4261 gtcgtgaggt accggatcct ctagagtcga ctccggaata acttcgtata ggatacttta
     4321 tacgaagtta tgcagaatgg tagctggatt gtagctgcta ttagcaatat gaaacctctt
     4381 aataacttcg tatagcatac attatacgaa gttatggcgc gccctattac ttgtacagct
     4441 cgtccatgcc gccggtggag tggcggccct cggcgcgttc gtactgttcc acgatggtgt
     4501 agtcctcgtt gtgggaggtg atgtccaact tgatgttgac gttgtaggcg ccgggcagct
     4561 gcacgggctt cttggccttg taggtggtct tgacctcagc gtcgtagtgg ccgccgtcct
     4621 tcagcttcag cctctgcttg atctcgccct tcagggcgcc gtcctcgggg tacatccgct
     4681 cggaggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg gaggggaagt
     4741 tggtgccgcg cagcttcacc ttgtagatga actcgccgtc ctgcagggag gagtcctggg
     4801 tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg aagccctcgg
     4861 ggaaggacag cttcaagtag tcggggatgt cggcggggtg cttcacgtag gccttggagc
     4921 cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg cagggggcca cccttggtca
     4981 ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg ccctcgatct
     5041 cgaactcgtg gccgttcacg gagccctcca tgtgcacctt gaagcgcatg aactccttga
     5101 tgatggccat gttatcctcc tcgcccttgc tcacggcggc cgctggcacg gctccggcct
     5161 cggcttcgtc ttcgacgaga gtctcgacct cgatctccgt tccgccgatg ttcagtttgg
     5221 tggttttgcg gatatctccg tgaatcaata tgtgctcgtg gatcaggacg cgcaggtagt
     5281 gtcccaacaa cccccaacaa tttttactca tcagatcaat aatcgtgtga cctacggtgg
     5341 agccatagac gctcaggacg ccaaaacctt cgggccccaa aatgaagaga attgggaaca
     5401 taccccagct cacgaaaaac agccatgcca tgccggtcac gacctggcgg caccgaccct
     5461 ttggcacagt atgataaccc tcgatatatg ctttggcggc gtgaaaaaat gtgttcgcgc
     5521 catagcacaa tccaagacaa aagaagatga ctttaacata gccggttgcc atggcgctgg
     5581 tagcccccca cacgatagtc ccgatgtctg agacaaggag tcccatggtt ctcctgctgt
     5641 agtcgttgct caggccggtg aggttgctca ggcggataag gatgacagga caagtgagca
     5701 gccactctgc atagcgcagc cactgcaccc ggtgtcctgt ggcaaggtag agcatagagg
     5761 gattcttaaa ctcaaaaaag aactcgagaa tcaccttaac catttcaatg gcgcacacat
     5821 agatctcctc ccagccgcat gtagatttcc aggtttggta ggcatagaac atcagcagca
     5881 aaatgctgaa tcctgctgca agccactgca ggacatttga cgcggtctga gcgccgttcg
     5941 tgccgcgaga ttcaatccat ccggcacagt aacattgatc ctcagggacc aggacggacc
     6001 cgttcaccac cacaggatta gtaacgaaca aaagttcgcg tccgacggca gacaaagcgc
     6061 cgccatagtc catggtggct agcataactt cgtataaagt atcctatacg aagttatttg
     6121 ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagaata acttcgtata
     6181 atgtatgcta tacgaagtta tgaattcgat atcaagctta tcgataatca acctctggat
     6241 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     6301 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     6361 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     6421 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     6481 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     6541 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     6601 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc
     6661 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     6721 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     6781 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga
     6841 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
     6901 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg
     6961 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga
     7021 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat
     7081 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg
     7141 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
     7201 gacggggttt caccatattg gccaggctgg tctccaactc c
//
