LOCUS       sequence-199299-        7074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Anion channelrhodopsin from Rhodomonas lens (RHODO) for expression 
            in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7074)
  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 7074)
  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-RlACR_477-EYFP
FEATURES             Location/Qualifiers
     source          1..7074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(127..129,130..843)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(169..190)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(430..449)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     777..798
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             873..914
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             924..941
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(964..981)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    970..1194
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1240..1668
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1327..1346)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1537..1558
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1677..1697)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1682..2011
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1862..1997
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1924..1943
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2078..2872
                     /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(2132..2151)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2742..2761
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3048..3169
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3085..3104)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3139..3158
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3218..3234)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3218..3234)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3231..3253)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3242..3258
                     /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)."
     promoter        complement(join(3266..3272,3273..3290,3291..3296))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3311..3332
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3449..3466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3620..4208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3700..3719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4379..5170,5171..5239))
                     /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     5002..5021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5240..5344)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5419..5438)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5610..5989
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5990..6193
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6144..6164
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6238..6257
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6238..6256
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gatagagagt tcgccaagaa tgccgacaag gacggagaga tgaaggccat cgaggaagga
       61 gctgctggac ccgccgctga cgccaagaag gacgaggata tcaagctgtt cggcaaggcg
      121 gccgccgtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
      181 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
      241 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
      301 accctcgtga ccaccttcgg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg
      361 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
      421 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
      481 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
      541 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
      601 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
      661 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
      721 cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
      781 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
      841 taacatatgc tctagagggc ccgcggttcg aaggtaagcc tatccctaac cctctcctcg
      901 gtctcgattc tacgcgtacc ggtcatcatc accatcacca ttgagtttaa acccgctgat
      961 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     1021 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     1081 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     1141 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     1201 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     1261 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1321 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1381 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1441 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1501 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1561 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1621 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     1681 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     1741 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     1801 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     1861 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     1921 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     1981 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     2041 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     2101 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     2161 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     2221 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     2281 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     2341 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     2401 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     2461 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     2521 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     2581 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     2641 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     2701 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     2761 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     2821 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     2881 ctggggttcg cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     2941 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3001 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     3061 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3121 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3181 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3241 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3301 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3361 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3421 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3481 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3541 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3601 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3661 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3721 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3781 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3841 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3901 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3961 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4021 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4081 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4141 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4201 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4261 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4321 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4381 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4441 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4501 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4561 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4621 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4681 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4741 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4801 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4861 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4921 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4981 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5041 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5101 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5161 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5221 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5281 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5341 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     5401 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     5461 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     5521 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     5581 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     5641 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     5701 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     5761 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     5821 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     5881 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     5941 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     6001 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     6061 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     6121 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     6181 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     6241 tacgactcac tatagggaga cccaagctgg ctagttaagc ttggtaccga gctcggatcc
     6301 atggccctgt acacctccca gtttggcatc gcctacggca gctgggaggc cgagaacgac
     6361 gattgcctga gcatgggcat cgctagcgga gcccccgtga agatcatcca gctgtgttgg
     6421 gccgtggtga gcctgtgcca ggtgattttc atgctcacaa gggcccccag ggtggcttgg
     6481 gaagccatct acctgcccct gtgcgagacc atcacctact tcctggcctt cagcggcaac
     6541 ggctacctga ggatggccga cggccagatc ttcccttggg ccaggatggc cagctggctg
     6601 gtgacctgcc ctgtgatgct gggccaggtg agcgccatgg ccctgatcaa gtacaaaagc
     6661 ctgcccctga acaatatgat ggtggccgcc tccctgatca gggtggtgat gggcatctcc
     6721 gccaccatca cacccaacga gcagctgaag tggatgttct tctttttcgg cgtgagcatg
     6781 ctgacctttg agtatttctg cgtgttcacc atcttcagca tcaccatcgc cgacttccag
     6841 gagatcgaca cacccaggtc caggttcgtg gtcggcaggc tgaagatcct caggattgtg
     6901 ttccactccg cttggacagc cttccctatc ctgtgggtgg tgtccagcac cggcctgtgc
     6961 gtgctcagcg agaatatctc cagcgtgtgc tacatgctcg ccgacgccgc ttgtaaaaat
     7021 ggctacggac tggtcctgtg gaacaccacc tggggccacc tgaacggcaa gtgg
//
