LOCUS       sequence-199325-        7071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Anion channelrhodopsin from Rhodomonas sp. (CCAC1630) for expression
            in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7071)
  AUTHORS   Govorunova EG, Sineshchekov OA, Rodarte EM, Janz R, Morelle O, 
            Melkonian M, Wong GK, Spudich JL
  TITLE     The Expanding Family of Natural Anion Channelrhodopsins Reveals 
            Large Variations in Kinetics, Conductance, and Spectral Sensitivity.
  JOURNAL   Sci Rep. 2017 Mar 3;7:43358. doi: 10.1038/srep43358.
  PUBMED    28256618
REFERENCE   2  (bases 1 to 7071)
  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: "2017-03-3"; volume: "7:43358. doi"; pages: " 
            10.1038/srep43358"
COMMENT     Sequence Label: pcDNA3.1-R2ACR_853-EYFP
FEATURES             Location/Qualifiers
     source          1..7071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     193..213
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(222..650)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(332..353)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     544..563
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    696..920
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     909..926
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(949..966)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(976..1017)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(join(1047..1760,1761..1763))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1092..1113)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1441..1460
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1700..1721
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2585..2594
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2704..2723)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2705..2723)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2768..2971)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2797..2817)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2972..3351
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3523..3542
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3617..3721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3722..3790,3791..4582)
                     /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(3940..3959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4753..5341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5242..5261
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5495..5512
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5629..5650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5665..5670,5671..5688,5689..5695)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5703..5719
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5708..5730
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5727..5743
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5727..5743
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5792..5913
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5803..5822)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5857..5876
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(6089..6883)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6200..6219)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6810..6829
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6950..7071,1..208))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6964..7071,1..28)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7018..7037)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
       61 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
      121 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
      181 gggactttcc acaccctaac tgacacacat tccacagaat taattcgcgt taaatttttg
      241 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
      301 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
      361 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
      421 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
      481 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
      541 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
      601 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc
      661 cctagagccc cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc
      721 atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
      781 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
      841 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
      901 ctggcaacta gaaggcacag tcgaggctga tcagcgggtt taaactcaat ggtgatggtg
      961 atgatgaccg gtacgcgtag aatcgagacc gaggagaggg ttagggatag gcttaccttc
     1021 gaaccgcggg ccctctagag catatgttac ttgtacagct cgtccatgcc gagagtgatc
     1081 ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg
     1141 ctcagggcgg actggtagct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg
     1201 atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg
     1261 cggatcttga agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg
     1321 tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg
     1381 atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg
     1441 gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc
     1501 atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcgggc gaagcactgc
     1561 aggccgtagc cgaaggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg
     1621 cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg
     1681 ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg
     1741 aacagctcct cgcccttgct cacggcggcc gcagcctctg tctcctcgct ggccttgccc
     1801 ctccagctat ccagggccat atcgatgtag gtctcgtgca cttcgttttt ctgaggcagc
     1861 ttgctggact ccagggtttc gggggtccag ctgccgttga ggacctggaa cagggtattg
     1921 gcgttgatga tgccgtacat gttcttgcac agcatatcag cgatcaggta ggccagcacg
     1981 gtggcgttct cattgatcat gcagatgccg gtgggggcca gtgtccagat gatggggaag
     2041 ctcacccagg cgaagaagaa caggctcctc atgaacatca gcctgttgta cacgatgttg
     2101 ttcagcttgg acctgaaggc gccaaacttc ttcaggccgg cggcgaaaat gctgctgacc
     2161 agaaagaagt cgaaggccag gcagatgcag cccaggaaga acaggaacca cttcatgaag
     2221 ttgttgctga tcacgctggc gccaatgccg tacacggtct ggatcagggt ggcggccatc
     2281 accatattct tgcagctcac gcccatccac ttcagctcat ggatgcccac cacctggaac
     2341 agcatgatgg gacaggtgat cagccagctg cagaatctgg cgatggggac caccctgccg
     2401 tcgctgagtc tcatgttgcc gttgccggtg gcggccacgc tgtacagaat ggtctccacc
     2461 aggggcaggt acacggcctc ccatgtgact ctgggggccc tgctggccat gaacaccacc
     2521 tgacaggcgc agatgcaggc gaacagcagc tggaagatct tggtggtgga ggtaatggtg
     2581 gggcccatgg cggcgcaatc catgttggag gcctcccagg tgccgtacag gccaaactgg
     2641 ggatcggcca cggcgctcat ggatccgagc tcggtaccaa gcttaactag ccagcttggg
     2701 tctccctata gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta
     2761 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     2821 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     2881 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     2941 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     3001 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     3061 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     3121 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     3181 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     3241 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     3301 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat
     3361 atctggcccg tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc
     3421 aattgtcggt caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga
     3481 cctccaacac acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg
     3541 tactgagagt gcaccatagg ggatcgggag atctcccgat ccgtcgacgt caggtggcac
     3601 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3661 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3721 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3781 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3841 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3901 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3961 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4021 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4081 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4141 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4201 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4261 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4321 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4381 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4441 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4501 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4561 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4621 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4681 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4741 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4801 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4861 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4921 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4981 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5041 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5101 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5161 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5221 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5281 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5341 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5401 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5461 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5521 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5581 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5641 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5701 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     5761 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     5821 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     5881 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     5941 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     6001 ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc
     6061 ggtcatttcg cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg
     6121 cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt
     6181 cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg
     6241 ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat
     6301 tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct
     6361 tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct
     6421 gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt
     6481 ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga
     6541 tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc
     6601 ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa
     6661 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac
     6721 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg
     6781 cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc
     6841 aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc
     6901 ctgtctcttg atcagatccg aaaatggata tacaagctcc cgggagcttt ttgcaaaagc
     6961 ctaggcctcc aaaaaagcct cctcactact tctggaatag ctcagaggca gaggcggcct
     7021 cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg g
//
