LOCUS       Exported                7203 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Anion channelrhodopsin from Geminigera cryophila (CCMP2564) for 
            expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1-GcACR_439-EYFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7203)
  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 7203)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7203
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             44..61
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(84..101)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    90..314
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      360..788
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(447..466)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     657..678
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(797..817)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        802..1131
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      982..1117
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1044..1063
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1198..1992
                     /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(1252..1271)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1862..1881
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2168..2289
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2205..2224)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2259..2278
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2338..2354)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2338..2354)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2351..2373)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2362..2378
                     /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(2386..2416)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2431..2452
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2569..2586)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2740..3328)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2820..2839)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3499..4359)
                     /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     4122..4141
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4360..4464)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4539..4558)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4730..5109
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5110..5313
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5264..5284
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5358..5377
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5358..5376
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6450..7166
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6492..6513)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6753..6772)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7100..7121
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7196..34
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
ORIGIN
        1 tatccctaac cctctcctcg gtctcgattc tacgcgtacc ggtcatcatc accatcacca
       61 ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
      121 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
      181 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
      241 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
      301 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
      361 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
      421 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
      481 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
      541 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
      601 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
      661 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
      721 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
      781 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
      841 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
      901 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
      961 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     1021 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     1081 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     1141 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     1201 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     1261 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     1321 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     1381 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     1441 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     1501 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     1561 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     1621 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     1681 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     1741 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     1801 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     1861 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     1921 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     1981 gagttcttct gagcgggact ctggggttcg cgaaatgacc gaccaagcga cgcccaacct
     2041 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     2101 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     2161 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2221 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2281 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     2341 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2401 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     2461 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2521 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2581 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2641 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2701 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2761 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2821 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2881 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2941 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3001 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3061 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3121 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3181 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3241 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3301 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3361 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3421 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3481 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3541 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3601 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3661 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3721 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3781 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3841 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3901 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3961 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4021 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4081 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4141 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4201 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4261 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4321 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4381 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4441 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     4501 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
     4561 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
     4621 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
     4681 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
     4741 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     4801 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     4861 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     4921 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     4981 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     5041 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     5101 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     5161 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     5221 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     5281 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
     5341 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagttaagc
     5401 ttggtaccga gctcggatcc atggccgaga ccggcgtgat cagaagccag gaagactact
     5461 acaggctcgt ggagaacagg gagacaatcg gcacatactg ggattcctcc ctggtgtacg
     5521 gcgagtgggc caggaacaac ggcaactgcc tcagacagtc cgaggtggac gtgcagttcg
     5581 tgaagatcct ccagctctgt atgagcgtgg tcagcagctg ccaggtgctg ttcatggtga
     5641 gcagagctcc tctggtgccc tgggaggcca tctacctccc catgaccgag agcattacct
     5701 atggcctggc cttcaccggc aacggctaca tcagactggc tacaggcaag atcctgccct
     5761 gggccaggat ggccagctgg ctgtgcacct gccccatcat gctgggcatg gtgtccaata
     5821 tggccctggt caagtacaag agccagcccc tgaaccccct gatggtggcc agctccatca
     5881 tcagggtggt gttcggcatc tccgccacaa tgagcgacga cagccagccc gccaaatggg
     5941 tgttcttctt catcgccgtg attttcttcc tcttcgagct gacctgcgtg tacgctatct
     6001 tcggaatgac catcgccgac tttgcccaaa tccagtcccc cctgggcgat gccgtggtct
     6061 acaggctgaa catcctgagg ggcatctttt tcgccagctg gacatgcttt cccatcctgt
     6121 ggctggtgtc ctccaccagc gcctgcctgc tggacgagaa tatcagcgcc gtgctgtacc
     6181 tgatggccga cgccctctgt aagaacagct acggcattgt cctgtggagc acaacctggg
     6241 gcctgctgaa cggcaagtgg gacagagagt atcccagaaa cagggacatc gacggcctga
     6301 tgatggacat cgaccccgcc aaggagaagc aactggagga gatgcccaac cagaacttcg
     6361 acatcaagat catgggccag accatcgctt ccgtgaagag aagctgcaga aggcctagag
     6421 ccgatatgga gagccaggat gcggccgccg tgagcaaggg cgaggagctg ttcaccgggg
     6481 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     6541 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     6601 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgcagtgct
     6661 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     6721 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     6781 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     6841 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     6901 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     6961 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     7021 gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc
     7081 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     7141 tcggcatgga cgagctgtac aagtaacata tgctctagag ggcccgcggt tcgaaggtaa
     7201 gcc
//
