LOCUS       Exported                7479 bp ds-DNA     circular SYN 13-MAY-2022
DEFINITION  Optogenetics.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a DIO-rsChRmine-oScarlet-Kv2.1-WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7479)
  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 7479)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7479
                     /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(1765..2457)
                     /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(2040..2059)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2309..2327
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3450..3459
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(3464..3497)
                     /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(3548..3581)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3606..4194
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3659..3679)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4077..4088)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4226..4702
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4425..4444)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4742..4882
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4742..4871
                     /label=AAV2 ITR
     rep_origin      4957..5412
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5044..5063)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5254..5275
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5429..5448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5548..5570
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5608..5626)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5694..5798
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5799..6659
                     /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(6017..6036)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6830..7418
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7319..7338
                     /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 gtagcagaag aatctggtgg cttcagggaa gtccgtggca cagctgatga agctgtcaat
     1621 gctggacatg ctccgcatgt cgatgacgcc ctccgtgcgt gcgggcagag gggccacggg
     1681 gctgggcatg ctgctcatct ccagctcctc tggaggcttg ctctgcgggg ccatctcctt
     1741 ggtgttgagg atgggctggg actgcttgta cagctcgtcc atgccgccgg tggagtggcg
     1801 gccctcggag cgttcgtact gttccaccac ggtgtagtcc tcgttgtggg aggtgatgtc
     1861 caacttgcgg tccacgttgt aggcgccggg catctgcacg ggcttcttgg ccttgtaggt
     1921 ggtcttgaag tccgccaggt agcgcccgcc gtccttcagg cgcagggcca tcttaatgtc
     1981 gcccttcagc acgccgtcct cggggtacaa ccgctcggtg gacgcttccc agcccattgt
     2041 cttcttctgc attacggggc cgtcaggagg gaagttggtg ccgcggagct tcaccttgta
     2101 gatcagggtg ccgtcctcca gggaggtgtc ctgggtcacg gtcacggcgc cgccgtcctc
     2161 gaagttcatc acgcggtccc acttgaagcc ctcggggaag gactgcttat agtagtcggg
     2221 gatgtcggcg gggtgcttgg tgaaggccct ggagccgtac atgaactgag gggacaggat
     2281 gtcccaggag aagggcaggg ggccaccctt ggtcaccttc agcttggcgg tctgggtgcc
     2341 ctcgtagggg cggccctcgc cctcgccctc gatctcgaac tcgtggccgt tcatggagcc
     2401 ctccatgtgc accttgaacc gcatgaactc cttgatcact gcctcgccct tgctcaccac
     2461 gttgatgtcg atctggtcca gggggatgta ctcgccctcg ctggtgatcc tgctcttggc
     2521 ggccgcagac agtctccgca ggcgctcgcc attgctgctc agcctcatat ccagctccag
     2581 cttctggaac tcatcctcgt cgtgtcccag ctgctccagc agccgaaaca gctcctcatc
     2641 ataggtcttt ctgaaccggg ccagggcaaa gccgtaacag gacttggcca cgatgtcgca
     2701 cacgcagtgg gccacctctg tccagttatc gtcgatccag ccgaagcctc tagggcagat
     2761 ggcccacacc aggggaaaca gaatccacag catggagaag gtaaacacgg cgaacttcag
     2821 cacgtgcagg ctgtgctcgg cgcctgtatc ctgggccagc tgggccagcc ggctatactg
     2881 cttggccacg atgctcatca cgatagagta ggcgaagata aaccagaaac agctaaagcc
     2941 ataccaggcg taggcgccat tcctcagcct agggtcgccc tctgcgtaga actcggccag
     3001 cacgccagac atcagcatgg caaagatgat ggcgctacaa gacacgcgat agggggccct
     3061 cagctgatac agcagatcgt acaccagcat tgggcaggtc agcatgtagt cggcatacct
     3121 gtaattgccg aagccgatga tgaaggggcc cacatcgccg aagttgtggc ccagtgtgtc
     3181 ggccacgatc ttgccgaagt agctcagcat ggcaaagaaa ttcacaaaca gggccaggtt
     3241 ctgctcctcc tgatagccgg cgtaaaagga ggttctcttg ctagggcccc gagatgtcca
     3301 ggactcatag cccaggtaga atgtggtgat tgtcagcacg ataaagcaag accagtgggc
     3361 gccgatggcc acggagttca gcttggtatc cagataccag ccgtccattg tgcccacgta
     3421 gaacatctgg tcggtgcctg gtgcgtgtgc catggtggct agcataactt cgtataaagt
     3481 atcctatacg aagttatttg ccttaaccca gaaattatca ctgttattct ttagaatggt
     3541 gcaaagaata acttcgtata atgtatgcta tacgaagtta tgaattcgat atcaagctta
     3601 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     3661 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     3721 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     3781 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     3841 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     3901 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     3961 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     4021 tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     4081 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     4141 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     4201 accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct
     4261 cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg
     4321 catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg
     4381 gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag
     4441 ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat
     4501 tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta
     4561 atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc
     4621 ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac
     4681 cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg
     4741 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     4801 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     4861 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     4921 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     4981 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     5041 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5101 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5161 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5221 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5281 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     5341 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5401 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5461 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5521 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5581 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     5641 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5701 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5761 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5821 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5881 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5941 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6001 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6061 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6121 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6181 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6241 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6301 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6361 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6421 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6481 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6541 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6601 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6661 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6721 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6781 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6841 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6901 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6961 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7021 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7081 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7141 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7201 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7261 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7321 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7381 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7441 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
