LOCUS       Exported                7008 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Anion channelrhodopsin from Rhodomonas salina (CCMP1319) for 
            expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1-RsACR_995-EYFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7008)
  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 7008)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7008
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      500..1088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     989..1008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1242..1259
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1376..1397
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1412..1442
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1450..1466
                     /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     1455..1477
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1474..1490
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1474..1490
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1539..1660
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1550..1569)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1604..1623
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1836..2630)
                     /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(1947..1966)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2557..2576
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2697..3026)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2711..2846
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2765..2784)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3011..3031
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3040..3468)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3150..3171)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3362..3381
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3514..3738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3727..3744
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3767..3784)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3794..3835)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(3865..4581)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3910..3931)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4259..4278
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4518..4539
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5459..5478)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5460..5478)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5523..5726)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5552..5572)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5727..6106
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6278..6297
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6372..6476
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6477..329
                     /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(6695..6714)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
       61 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      121 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      181 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      241 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      301 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
      361 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
      421 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
      481 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
      541 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
      601 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
      661 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      721 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      781 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
      841 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      901 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      961 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1021 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1081 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1141 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1201 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1261 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1321 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     1381 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     1441 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     1501 ccaagctcta gctagaggtc gacggtatac agacatgata agatacattg atgagtttgg
     1561 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     1621 tgctttattt gtaaccatta taagctgcaa taaacaagtt ggggtgggcg aagaactcca
     1681 gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg attccgaagc
     1741 ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg
     1801 cttggtcggt catttcgcga accccagagt cccgctcaga agaactcgtc aagaaggcga
     1861 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     1921 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     1981 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     2041 atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg
     2101 cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     2161 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     2221 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     2281 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     2341 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     2401 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc
     2461 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg
     2521 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     2581 tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat
     2641 cctcatcctg tctcttgatc agatccgaaa atggatatac aagctcccgg gagctttttg
     2701 caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggcagag
     2761 gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg
     2821 gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac
     2881 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     2941 ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     3001 gactttccac accctaactg acacacattc cacagaatta attcgcgtta aatttttgtt
     3061 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     3121 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     3181 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     3241 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     3301 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     3361 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     3421 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg ggataccccc
     3481 tagagcccca gctggttctt tccgcctcag aagccataga gcccaccgca tccccagcat
     3541 gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca ccccccagaa
     3601 tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag gaaaggacag
     3661 tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca acagatggct
     3721 ggcaactaga aggcacagtc gaggctgatc agcgggttta aactcaatgg tgatggtgat
     3781 gatgaccggt acgcgtagaa tcgagaccga ggagagggtt agggataggc ttaccttcga
     3841 accgcgggcc ctctagagca tatgttactt gtacagctcg tccatgccga gagtgatccc
     3901 ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct
     3961 cagggcggac tggtagctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat
     4021 gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg
     4081 gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agacgttgtg
     4141 gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat
     4201 gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt
     4261 cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat
     4321 ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcgggcga agcactgcag
     4381 gccgtagccg aaggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca
     4441 gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct
     4501 gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa
     4561 cagctcctcg cccttgctca cggcggccgc cacgggcttg ccaaacagct tgatgtcctc
     4621 gtccttcttg gcgtcggcgg caggtccggc tgtgccctcc tcgatagctt tcatctcgcc
     4681 atccttgtcg gcgttcttgg cgaactccct gtcccacttg ccgttcagat ggccccatgt
     4741 tgtgttccac aggatcaggc cgtagccgtt cttgcaggcg gcatcggcca gcatgtagca
     4801 cacggagctg atgttctcgc tcagcacaca caggccggtg ctgctcacca cccagaggat
     4861 ggggaaggcg gtccaggcgc tgtggaacac gattctcagg atcttcagcc ttcccaccac
     4921 cagcctgctc ctgggggtgt cgatctcctg gaagtcggcg atggtgatgg agaagatggt
     4981 gaagacgcag aagtactcga aggtcagcat ggagacgccg aagaagaaaa acatccactt
     5041 cagctgctcg ttgggggtga ttgtggcgct gatgcccatc accaccctaa tcagggaggc
     5101 agccaccatc atgttgttca ggggcaggct cttgtacttg atcagggcca tggcggacac
     5161 ctggcccagc atcacagggc aggtcaccag ccaggaggcc atcctggccc aggggaagat
     5221 ctggccgtcg gccatcctga ggtagccgtt gccggtgaag gccaggaagt aggtgattgt
     5281 ctcgcacagg ggcagataga tggcctccca agccacccta ggggccctgg tcagcatgaa
     5341 gatcacctgg cacaggctga ccacggccca gcacagctgg ataatcttca cgggggcgcc
     5401 gctggcgatg cccatggatc cgagctcggt accaagctta actagccagc ttgggtctcc
     5461 ctatagtgag tcgtattaat ttcgataagc cagtaagcag tgggttctct agttagccag
     5521 agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg
     5581 gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca
     5641 ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca
     5701 ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac
     5761 tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta
     5821 ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag
     5881 tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc tattggcgtt
     5941 actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca
     6001 ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat
     6061 gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaacg cgtatatctg
     6121 gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt catgcaattg
     6181 tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc agcgacctcc
     6241 aacacacaag cagggagcag atactggctt aactatgcgg catcagagca gattgtactg
     6301 agagtgcacc ataggggatc gggagatctc ccgatccgtc gacgtcaggt ggcacttttc
     6361 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     6421 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     6481 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     6541 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6601 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     6661 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     6721 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6781 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6841 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6901 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6961 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgaca
//
