LOCUS       Exported                5767 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Slow cycling (open for minutes) step-function artificial anion 
            conducting channelrhodopsin (aACR). High light sensitivity. 
            Activation with blue to green light, inactivation max with 605 nm 
            (accelarates closure to ms). Codon optimized for mammalian 
            expression..
ACCESSION   .
VERSION     .
KEYWORDS    AAV2_hSyn_iChloC_CA_Citrine
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5767)
  AUTHORS   Wietek J, Rodriguez-Rozada S, Tutas J, Tenedini F, Grimm C, Oertner 
            TG, Soba P, Hegemann P, Wiegert JS
  TITLE     Anion-conducting channelrhodopsins with tuned spectra and modified 
            kinetics engineered for optogenetic manipulation of behavior.
  JOURNAL   Sci Rep. 2017 Nov 2;7(1):14957. doi: 10.1038/s41598-017-14330-y.
  PUBMED    29097684
REFERENCE   2  (bases 1 to 5767)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5767
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   33..162
                     /label=AAV2 ITR
     promoter        244..691
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      709..718
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             715..1641
                     /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
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEQIYVCAIRMVKVILEFFFSFKN
                     PSMLYLATGHRVQWLRYAEWLLTAPVILIHLSNLTGLSNDYSRRTMGLLVSNIGCIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     CDS             1654..2370
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1699..1720)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1960..1979)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2307..2328
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2386..2974
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2439..2459)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2857..2868)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2977..2993)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2978..2994)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3023..3144
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3034..3053)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3088..3107
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3352..3369)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3523..4111)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3603..3622)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4282..5142)
                     /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     4905..4924
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5143..5247)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5273..5728)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5410..5431)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5622..5641
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gatcggtgcg ggcctcttcg ctattacgcc agctgcgcgc tcgctcgctc actgaggccg
       61 cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
      121 cgcgcagaga gggagtggcc aactccatca ctaggggttc cttgtagtta atgattaacc
      181 cgccatgcta cttatctacg tagccatgct ctaggaagat ctctgcagag ggccctgcgt
      241 atgagtgcaa gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac
      301 cgaccccgac ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc
      361 agagaggggg aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc
      421 gcggacagtg ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg
      481 cgcgctgacg tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt
      541 ccgcgccgcc gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg
      601 gcgcgaccat ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag
      661 cggaggagtc gtgtcgtgcc tgagagcgca gtcgaattca agctgctagc caccatggac
      721 tatggcggcg ctttgtctgc cgtcggacgc gaacttttgt tcgttactaa tcctgtggtg
      781 gtgaacgggt ccgtcctggt ccctgaggat caatgttact gtgccggatg gattgaatct
      841 cgcggcacga acggcgctca gaccgcgtca aatgtcctgc agtggcttgc agcaggattc
      901 agcattttgc tgctgatgtt ctatgcctac caaacctgga aatctacatg cggctgggag
      961 cagatctatg tgtgcgccat tagaatggtt aaggtgattc tcgagttctt ttttagcttt
     1021 aagaatccct ctatgctcta ccttgccaca ggacaccggg tgcagtggct gcgctatgca
     1081 gagtggctgc tcactgctcc tgtcatcctt atccacctga gcaacctcac cggcctgagc
     1141 aacgactaca gcaggagaac catgggactc cttgtctcaa acatcgggtg tatcgtgtgg
     1201 ggggctacca gcgccatggc aaccggctat gttaaagtca tcttcttttg tcttggattg
     1261 tgctatggcg cgaacacatt ttttcacgcc gccaaagcat atatcgaggg ttatcatact
     1321 gtgccaaagg gtcggtgccg ccaggtcgtg accggcatgg catggctgtt tttcgtgagc
     1381 tggggtatgt tcccaattct cttcattttg gggcccgaag gttttggagt cctgagcgtc
     1441 tatggctcca ccgtaggtca cacgattatt gatctgatga gtaaaaattg ttgggggttg
     1501 ttgggacact acctgcgcgt cctgatccac gagcacatat tgattcacgg agatatccgc
     1561 aaaaccacca aactgaacat cggcggaacg gagatcgagg tcgagactct cgtcgaagac
     1621 gaagccgagg ccggagccgt ggcggcactc gagatggtga gcaagggcga ggagctgttc
     1681 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1741 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1801 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg
     1861 atgtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1921 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1981 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2041 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2101 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2161 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2221 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc
     2281 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2341 atcactctcg gcatggacga gctgtacaag tagaagctta tcgataatca acctctggat
     2401 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     2461 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     2521 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     2581 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     2641 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     2701 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     2761 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     2821 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     2881 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     2941 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc tcgacccggg
     3001 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     3061 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3121 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     3181 cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     3241 gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa
     3301 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3361 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3421 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3481 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3541 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3601 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3661 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3721 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3781 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3841 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3901 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3961 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4021 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4081 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4141 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4201 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4261 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4321 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4381 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4441 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4501 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4561 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4621 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4681 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4741 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4801 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4861 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4921 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4981 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5041 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5101 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5161 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5221 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     5281 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     5341 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     5401 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     5461 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     5521 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     5581 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     5641 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     5701 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg
     5761 gaagggc
//
