LOCUS       Exported                6653 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expression of a chimeric G-protein coupled receptor 
            (Rhodopsin-GPR173; Opto-GPR173; E12).
ACCESSION   .
VERSION     .
KEYWORDS    Opto-GPR173
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6653)
  AUTHORS   Morri M, Sanchez-Romero I, Tichy AM, Kainrath S, Gerrard EJ, 
            Hirschfeld PP, Schwarz J, Janovjak H
  TITLE     Optical functionalization of human Class A orphan G-protein-coupled 
            receptors.
  JOURNAL   Nat Commun. 2018 May 16;9(1):1950. doi: 10.1038/s41467-018-04342-1.
  PUBMED    29769519
REFERENCE   2  (bases 1 to 6653)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6653
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        74..453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        454..657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     608..628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     702..721
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        702..720
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             813..845
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     CDS             870..908
                     /codon_start=1
                     /product="segment of bovine rhodopsin containing a signal 
                     for N-linked glycosylation (Favaloro et al., 2008)"
                     /label=opsin tag
                     /note="The asparagine in the NKT tripeptide will be 
                     glycosylated if this sequence is exposed to the ER lumen."
                     /translation="GPNFYVPFSNKTG"
     primer_bind     complement(2083..2100)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2089..2313
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2359..2787
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2446..2465)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2656..2677
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2796..2816)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2801..3130
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2981..3116
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3043..3062
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3197..3991
                     /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(3251..3270)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3861..3880
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4165..4286
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4202..4221)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4256..4275
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4335..4351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4335..4351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4348..4370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4359..4375
                     /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(4383..4413)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4428..4449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4566..4583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4737..5325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4817..4836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5496..6356)
                     /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     6119..6138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6357..6461)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6536..6555)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
       61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      661 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
      721 gagacccaag ctggctagcg tttaaactta agcttgccac catgaaaacg atcatcgccc
      781 tgagctacat cttctgcctg gtattcgcca tgtacaccga tatagagatg aacaggctgg
      841 gaaaggatag cctcatgaac gggaccgagg gcccaaactt ctacgtgcct ttctccaaca
      901 agacgggcgt ggtgcgcagc cccttcgagg ccccgcagta ctacctggcg gagccatggc
      961 agttctccat gctggccgcc tacatgttcc tgctgatcat gcttggcttc cccatcaact
     1021 tcctcacgct gtacgtcgtg ctcaaggagc gtgccctgca caaggctctc aactacatcc
     1081 tgctcaacct ggccgtggcc gacctcttca tggtgttcgg gggcttcacc accaccctct
     1141 acacctctct gcacgggtac ttcgtctttg ggcccacggg ctgcaacctg gagggcttct
     1201 ttgccacctt gggcggtgaa attgcactgt ggtccttggt ggtcctggcc atcgagcggt
     1261 acgtggtggt ggcccaccac cgcttctacg ccaagcgcat gacactctgg acagccatca
     1321 tgggcgtcgc cttcacctgg gtcatggctc tggcctgtgc cgcgcccccc ctcgtcggct
     1381 ggtccaggta catcccggag ggcatgcagt gctcgtgcgg gattgactac tacacgcccc
     1441 acgaggaaac caacaatgag tcgttcgtca tctacatgtt cgtggtccac ttcatcatcc
     1501 ccctgattgt catattcttc tgctacggat accgtcaccg caagatgaag ccagtgcaga
     1561 tggtgccagc catcagccag aactggacat tccatggtcc cggggccacc ggccaggctg
     1621 ctgccaactg gatcgccggc tttggccgtg ggcccatgcc accaaccctg ctgggtatcc
     1681 ggcagaatgg gcatgcagcc agccggcggc tactgggcat ggacgaggtc aagggtgaaa
     1741 agcagctggg ccgcatggtg atcatcatgg tcatcgcttt cctaatctgc tggctgccct
     1801 acgctggggt ggcgttctac atcttcaccc atcagggctc tgactttggc cccatcttca
     1861 tgaccatccc ggctttcttt gccaagactt ctgccgtcta caaccccgtc atctacatca
     1921 tgatgaacaa gcagttccgc aagtgcctga ggactcacgc cccctgctgg ggcacaggag
     1981 gtgccccggc tcccagagaa ccctactgtg tcatgacaga aaccagccaa gtggcgcctg
     2041 cctaactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     2101 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2161 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2221 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2281 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     2341 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2401 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2461 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2521 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2581 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2641 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2701 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2761 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     2821 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2881 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2941 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3001 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3061 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3121 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     3181 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     3241 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     3301 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3361 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3421 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3481 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3541 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3601 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3661 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3721 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3781 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3841 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     3901 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     3961 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4021 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4081 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4141 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4201 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4261 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4321 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4381 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4441 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4501 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4561 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4621 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4681 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4741 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4801 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4861 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4921 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4981 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5041 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5101 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5161 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5221 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5281 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5341 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5401 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5461 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5521 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5581 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5641 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5701 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5761 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5821 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5881 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5941 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6001 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6061 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6121 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6181 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6241 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6301 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6361 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6421 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6481 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     6541 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     6601 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gac
//
