LOCUS       Exported                7163 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV-mediated expression of ChrimsonR-GFP under the EF1-alpha 
            promoter (1.1kb short version), in floxed/reversed (Cre-dependent) 
            manner. Using bGHpA signal..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-EF1-alpha-1.1-FLEX-rc [ChrimsonR-GFP]
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7163)
  AUTHORS   Klapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK,
            Morimoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, 
            Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, 
            Constantine-Paton M, Wong GK, Boyden ES
  TITLE     Independent optical excitation of distinct neural populations.
  JOURNAL   Nat Methods. 2014 Mar;11(3):338-46. doi: 10.1038/nmeth.2836. Epub 
            2014 Feb 9.
  PUBMED    24509633
REFERENCE   2  (bases 1 to 7163)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7163
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        203..1381
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          434..1372
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1329..1349
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1390..1423)
                     /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(1486..1519)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1536..2249)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1578..1599)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1927..1946
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2186..2207
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2268..3317)
                     /codon_start=1
                     /product="red light-drivable channelrhodopsin from 
                     Chlamydomonas noctigama (Klapoetke et al., 2014)"
                     /label=ChrimsonR
                     /note="K176R mutant with accelerated closing kinetics"
                     /translation="MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTE
                     WWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSI
                     AIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRY
                     FEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIV
                     SCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLK
                     LSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEE
                     FVEEEDEDTV"
     regulatory      3314..3323
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    3345..3378
                     /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    3449..3482
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3515..4103
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3568..3588)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3986..3997)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4144..4351
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4521..4650
                     /label=AAV2 ITR
     primer_bind     complement(4716..4733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4887..5475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4967..4986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5646..6506)
                     /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     6269..6288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6507..6611)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6637..7092)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6774..6795)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6986..7005
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagatcg taggttaatt aaggctccgg tgcccgtcag tgggcagagc gcacatcgcc
      241 cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg
      301 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
      361 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
      421 cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta
      481 tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga
      541 gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc
      601 tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca
      661 ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc
      721 tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac
      781 tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg
      841 ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc
      901 tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc
      961 aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc
     1021 tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac
     1081 acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg
     1141 ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg
     1201 gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag
     1261 gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg
     1321 gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg
     1381 agaattccca taacttcgta taaagtatcc tatacgaagt tatatcaaaa taggaagacc
     1441 aatgcttcac catcgacccg aattgccaag catcaccatc gacccataac ttcgtataat
     1501 gtatgctata cgaagttata ctagctagct cattacttgt acagctcgtc catgccgaga
     1561 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg
     1621 tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag cacggggccg
     1681 tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg
     1741 ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag
     1801 acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg
     1861 aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg
     1921 gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct
     1981 tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag
     2041 cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg
     2101 gtggtgcaaa tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg
     2161 gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc
     2221 ccggtgaaca gctcctcgcc cttgctcact gctactaccg gtgccgccac tgtgtcctcg
     2281 tcctcctcct ccacgaactc ttccacttcc acctcctcgc ctccgatctc catcttggtg
     2341 gtcttccgga tgtcgccgtg gatcaggatg tgctcgtgga tcttgatcct caggtggtgg
     2401 gccaggaagg tccaaaactc cttggcgatg atgtcgcaga tgctgtggcc gatgctgttg
     2461 gcgtaagggc tcagcttcag cagtccttct ggtcccactg cccagaggat tgggtagctg
     2521 ccccaagagg cgaagtaagc gtaggccatc agcttcacga ccatgcggca atggccttta
     2581 ggcacgctgt ggttggcttc cacgtagcac ttggcggcct ggaagtacat gtagccgccg
     2641 tagatgcaag acacgatata cagcagccac ttgagccaat cggtagccag tcctgcggcc
     2701 atgccgaaca cgatcattcc cacgcaagac acgatcaggc ccatggtccg cttgctgtag
     2761 tcgttcttca ggccgctcag gttgctcagt ctgatcagga tcacggggca agacagcagc
     2821 cactcgaagt agcgcaggca ataggcgtgg ttgccggtag acaggtacac tgtggcgggg
     2881 ctgctgaact ccttgaagat ctccagggtc acgaacagca cctcgacgca acagacgtag
     2941 acctcctccc aaccgcaagt ggccttccag gcgctgaagc cgtagaatgt cagcagggcg
     3001 atggcgatgc tgaaagcaat ccactggcag acctgggcgc cgatcttttc tcctggtgtg
     3061 cctctgctgt gcagccaagc ctgcttcttc tccactccca cgaccaggta gccgcctgtg
     3121 gcctccacga agcaatatcc gtatccggcg tcgctcagtc cgtaagaagg gtcacaccac
     3181 cactcggtgc tgaagcactc gcctgtaggt gtcattcctc cacagcccat gtcctcgtgg
     3241 tggtaggggt taggccaagg gttgatgcct ccggcggcaa acagagatct ggtggcgctg
     3301 ctgatcagct cagccatggt ggcggcgcgg cctaggtacc tagtataact tcgtatagga
     3361 tactttatac gaagttatca ttgggattct tcctattttg atccaagcat caccatcgac
     3421 cctctagtcc agatctcacc atcgacccat aacttcgtat agcatacatt atacgaagtt
     3481 atgtccctcg aagaggttcg cgaagcttat cgataatcaa cctctggatt acaaaatttg
     3541 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3601 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     3661 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     3721 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3781 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3841 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3901 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     3961 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     4021 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     4081 ctccctttgg gccgcctccc cgcatcgata ccgtcgaccc gggcggccgc tcataaagct
     4141 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4201 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4261 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4321 attgggaaga caatagcagg catgctgggg atgcggtccg gactgtacaa ttgcattcat
     4381 tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac
     4441 aaatgtggta aaatcgataa ggatcttcct agagcatggc tacgtagata agtagcatgg
     4501 cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg
     4561 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4621 ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat gaatcggcca acgcgcgggg
     4681 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4741 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4801 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4861 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4921 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4981 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5041 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5101 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5161 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5221 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5281 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5341 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5401 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5461 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5521 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5581 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5641 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5701 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5761 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5821 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5881 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5941 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6001 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6061 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6121 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6181 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6241 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6301 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6361 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6421 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6481 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6541 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6601 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     6661 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     6721 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     6781 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     6841 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     6901 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     6961 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     7021 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     7081 tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     7141 gggcctcttc gctattacgc cag
//
