LOCUS       Exported                7284 bp ds-DNA     circular SYN 18-MAY-2022
DEFINITION  Optogenetics.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a DIO-rsChRmine-oScarlet-WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7284)
  AUTHORS   Kishi KE, Kim YS, Fukuda M, Inoue M, Kusakizako T, Wang PY, 
            Ramakrishnan C, Byrne EFX, Thadhani E, Paggi JM, Matsui TE, 
            Yamashita K, Nagata T, Konno M, Quirin S, Lo M, Benster T, Uemura T,
            Liu K, Shibata M, Nomura N, Iwata S, Nureki O, Dror RO, Inoue K, 
            Deisseroth K, Kato HE
  TITLE     Structural basis for channel conduction in the pump-like 
            channelrhodopsin ChRmine.
  JOURNAL   Cell. 2022 Feb 17;185(4):672-689.e23. doi: 
            10.1016/j.cell.2022.01.007. Epub 2022 Feb 2.
  PUBMED    35114111
REFERENCE   2  (bases 1 to 7284)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 18, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7284
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1416..1449
                     /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    1500..1533
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1570..2262)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWDRVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(1845..1864)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2114..2132
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3255..3264
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(3269..3302)
                     /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(3353..3386)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3411..3999
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3464..3484)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3882..3893)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4031..4507
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4230..4249)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4547..4687
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4547..4676
                     /label=AAV2 ITR
     rep_origin      4762..5217
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4849..4868)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5059..5080
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5234..5253)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5353..5375
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5413..5431)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5499..5603
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5604..6464
                     /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(5822..5841)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6635..7223
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7124..7143
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gatccataac ttcgtatagg atactttata
     1441 cgaagttatg cagaatggta gctggattgt agctgctatt agcaatatga aacctcttaa
     1501 taacttcgta tagcatacat tatacgaagt tatggcgcgc cctacctaca cctcgttctc
     1561 gtagcagaac ttgtacagct cgtccatgcc gccggtggag tggcggccct cggagcgttc
     1621 gtactgttcc accacggtgt agtcctcgtt gtgggaggtg atgtccaact tgcggtccac
     1681 gttgtaggcg ccgggcatct gcacgggctt cttggccttg taggtggtct tgaagtccgc
     1741 caggtagcgc ccgccgtcct tcaggcgcag ggccatctta atgtcgccct tcagcacgcc
     1801 gtcctcgggg tacaaccgct cggtggacgc ttcccagccc attgtcttct tctgcattac
     1861 ggggccgtca ggagggaagt tggtgccgcg gagcttcacc ttgtagatca gggtgccgtc
     1921 ctccagggag gtgtcctggg tcacggtcac ggcgccgccg tcctcgaagt tcatcacgcg
     1981 gtcccacttg aagccctcgg ggaaggactg cttatagtag tcggggatgt cggcggggtg
     2041 cttggtgaag gccctggagc cgtacatgaa ctgaggggac aggatgtccc aggagaaggg
     2101 cagggggcca cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc
     2161 ctcgccctcg ccctcgatct cgaactcgtg gccgttcatg gagccctcca tgtgcacctt
     2221 gaaccgcatg aactccttga tcactgcctc gcccttgctc accacgttga tgtcgatctg
     2281 gtccaggggg atgtactcgc cctcgctggt gatcctgctc ttggcggccg cagacagtct
     2341 ccgcaggcgc tcgccattgc tgctcagcct catatccagc tccagcttct ggaactcatc
     2401 ctcgtcgtgt cccagctgct ccagcagccg aaacagctcc tcatcatagg tctttctgaa
     2461 ccgggccagg gcaaagccgt aacaggactt ggccacgatg tcgcacacgc agtgggccac
     2521 ctctgtccag ttatcgtcga tccagccgaa gcctctaggg cagatggccc acaccagggg
     2581 aaacagaatc cacagcatgg agaaggtaaa cacggcgaac ttcagcacgt gcaggctgtg
     2641 ctcggcgcct gtatcctggg ccagctgggc cagccggcta tactgcttgg ccacgatgct
     2701 catcacgata gagtaggcga agataaacca gaaacagcta aagccatacc aggcgtaggc
     2761 gccattcctc agcctagggt cgccctctgc gtagaactcg gccagcacgc cagacatcag
     2821 catggcaaag atgatggcgc tacaagacac gcgatagggg gccctcagct gatacagcag
     2881 atcgtacacc agcattgggc aggtcagcat gtagtcggca tacctgtaat tgccgaagcc
     2941 gatgatgaag gggcccacat cgccgaagtt gtggcccagt gtgtcggcca cgatcttgcc
     3001 gaagtagctc agcatggcaa agaaattcac aaacagggcc aggttctgct cctcctgata
     3061 gccggcgtaa aaggaggttc tcttgctagg gccccgagat gtccaggact catagcccag
     3121 gtagaatgtg gtgattgtca gcacgataaa gcaagaccag tgggcgccga tggccacgga
     3181 gttcagcttg gtatccagat accagccgtc cattgtgccc acgtagaaca tctggtcggt
     3241 gcctggtgcg tgtgccatgg tggctagcat aacttcgtat aaagtatcct atacgaagtt
     3301 atttgcctta acccagaaat tatcactgtt attctttaga atggtgcaaa gaataacttc
     3361 gtataatgta tgctatacga agttatgaat tcgatatcaa gcttatcgat aatcaacctc
     3421 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     3481 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     3541 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     3601 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     3661 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     3721 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     3781 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctatgttg
     3841 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     3901 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     3961 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg
     4021 agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
     4081 tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
     4141 ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
     4201 tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc
     4261 acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc
     4321 tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg
     4381 gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat
     4441 ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc
     4501 tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg
     4561 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4621 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4681 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4741 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4801 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     4861 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4921 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     4981 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5041 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     5101 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     5161 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     5221 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     5281 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5341 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5401 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     5461 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5521 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5581 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5641 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5701 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5761 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5821 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5881 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5941 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6001 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6061 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6121 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6181 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6241 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6301 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6361 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6421 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6481 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6541 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6601 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6661 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6721 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6781 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6841 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6901 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6961 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     7021 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     7081 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7141 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7201 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7261 tttgctggcc ttttgctcac atgt
//
