LOCUS       sequence-134333-        7291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An improved chloride-conducting channelrhodopsin for light-induced 
            inhibition of neuronal activity in vivo. Cre inducible expression 
            (double floxed inversed ORF).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7291)
  AUTHORS   Wietek J, Beltramo R, Scanziani M, Hegemann P, Oertner TG, Simon 
            Wiegert J
  TITLE     An improved chloride-conducting channelrhodopsin for light-induced 
            inhibition of neuronal activity in vivo.
  JOURNAL   Sci Rep. 2015 Oct 7;5:14807. doi: 10.1038/srep14807.
  PUBMED    26443033
REFERENCE   2  (bases 1 to 7291)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2015-10-7"; volume: "5:14807. doi"; pages: " 
            10.1038/srep14807"
COMMENT     Sequence Label: pAAV EF1a DIO iChloC 2A dsRed
FEATURES             Location/Qualifiers
     source          1..7291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1449..1482)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1533..1566)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1575..2255)
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MVASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGTLIYKVKFRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFL"
     primer_bind     complement(1637..1660)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1835..1854)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2052..2072
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2104..2122
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2248..2257
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2252..2261
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2277..2330)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(2340..3266)
                     /codon_start=1
                     /product="N-terminal fragment of channelrhodopsin-2 from 
                     the alga Chlamydomonas reinhardtii (Nagel et al., 2003)"
                     /label=ChR2
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEQIYVCAIRMVKVILEFFFSFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSNIGCIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3263..3272
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3277..3310
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3361..3394
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3419..4006
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3471..3491)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3889..3900)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4038..4514
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4237..4256)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4554..4694
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4554..4683
                     /label=AAV2 ITR
     rep_origin      4769..5224
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4856..4875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5066..5087
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5241..5260)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5360..5382
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5420..5438)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5506..5610
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5611..5679,5680..6471)
                     /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(5829..5848)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6642..7230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7131..7150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtacggatcc tctagagtcg
     1441 actccggaat aacttcgtat aggatacttt atacgaagtt atgcagaatg gtagctggat
     1501 tgtagctgct attagcaata tgaaacctct taataacttc gtatagcata cattatacga
     1561 agttatggcg cgccctacag gaacaggtgg tggcggccct cggagcgctc gtactgttcc
     1621 acgatggtgt agtcctcgtt gtgggaggtg atgtccagct tggtgtccac gtagtagtag
     1681 ccgggcagct gcacgggctt cttggccatg tagatggtct tgaactccac caggtagtgg
     1741 ccgccgtcct tcagcttcag ggcctggtgg atctcgccct tcagcacgcc gtcgcggggg
     1801 tacaggcgct cggtggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg
     1861 ggggggaagt tggtgccgcg gaacttcacc ttgtagatca gcgtgccgtc ctgcagggag
     1921 gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg
     1981 aagccctcgg ggaaggacag cttcttgtag tcggggatgt cggcggggtg cttcacgtac
     2041 gccttggagc cgtactggaa ctggggggac aggatgtccc aggcgaaggg cagggggccg
     2101 cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg
     2161 ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgcgcacctt gaagcgcatg
     2221 aactctttga tgacgtcctc ggaggaggcc accatggcgg ccgcggatcc cggtgcaggg
     2281 ccgggattct cctccacgtc accgcatgtt agaagacttc ctctgccctc ggccgcgggt
     2341 accgcgccag cctcggcctc gtcctccacc agcgtctcga cctcaatctc agtgccacca
     2401 atgttcaatt tggtggtctt gcgaatgtcg ccgtggatga ggatatgctc gtggatcagc
     2461 acgcgcaggt agtggccgag cagaccccag cagttcttcg acatcaggtc aatgatggtg
     2521 tggccgacgg tggagccgta cacgctcagg acgccgaagc cctcggggcc gaggatgaac
     2581 aggatgggga acatacccca tgatacgaag aagagccaag ccatgccagt caccacctgg
     2641 cgacaccggc ccttcggcac ggtgtggtaa ccctcgatgt aggccttggc agcgtgaaag
     2701 aacgtgttag caccataaca cagacccagg cagaagaaga tgaccttgac gtatccggtg
     2761 gccatggcgg aagtggcgcc ccacacaata cagccaatat tagacacaag cagacccatg
     2821 gtgcgcctgc tgtagtcgtt ggacaagccc gtcaggtttg acaggtgaat gagaatgacc
     2881 gggcaggtga gaagccactc ggcgtaacgc aaccactgga cgcggtggcc tgtggctaga
     2941 tacagcatgg acgggttctt aaacgagaag aagaactcga gaatcacctt gaccatcctg
     3001 atagcgcaca catagatctg ctcccagccg caggttgact tccatgtttg gtaggcgtaa
     3061 aacataagca gtaggatgga gaagccagca gcaagccatt gcagcacgtt cgacgccgtt
     3121 tgggcaccgt ttgtgccacg cgactcaatc cagcccgcgc agtaacactg gtcctcaggc
     3181 acaagtacag agccattgac gactactggg ttcgttacaa atagcagctc gcgcccaacg
     3241 gcactcaggg cgcctccata atccatggtg gctagcataa cttcgtataa agtatcctat
     3301 acgaagttat ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga
     3361 ataacttcgt ataatgtatg ctatacgaag ttatgaattc gatatcaagc ttatcgataa
     3421 tcaacctctg gatacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     3481 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3541 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3601 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3661 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3721 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3781 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3841 tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3901 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3961 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg
     4021 ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc
     4081 tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt
     4141 tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg
     4201 ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg
     4261 cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc
     4321 ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt
     4381 ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc
     4441 aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc
     4501 ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc
     4561 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4621 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     4681 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4741 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     4801 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4861 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4921 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4981 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5041 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5101 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5161 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     5221 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     5281 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5341 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5401 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     5461 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5521 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5581 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5641 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5701 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5761 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5821 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5881 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5941 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6001 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6061 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6121 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6181 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6241 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6301 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6361 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6421 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6481 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6541 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6601 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6661 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6721 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6781 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6841 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6901 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6961 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7021 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7081 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7141 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7201 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7261 ctggcctttt gctggccttt tgctcacatg t
//
