LOCUS Exported 6536 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expression of a chimeric G-protein coupled receptor (Rhodopsin-GPR18; Opto-GPR18; A8). ACCESSION . VERSION . KEYWORDS Opto-GPR18 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6536) 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 6536) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6536 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(46..65) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 237..616 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 617..820 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 771..791 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 865..884 /label=T7 /note="T7 promoter, forward primer" promoter 865..883 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 976..1008 /codon_start=1 /product="epitope tag from vesicular stomatitis virus G protein" /label=VSV-G tag /translation="YTDIEMNRLGK" CDS 1033..1071 /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(2129..2146) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2135..2359 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2405..2833 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2492..2511) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2702..2723 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2842..2862) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2847..3176 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3027..3162 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3089..3108 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3243..4037 /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(3297..3316) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3907..3926 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4211..4332 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4248..4267) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4302..4321 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4381..4397) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4381..4397) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4394..4416) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4405..4421 /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(4429..4459) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4474..4495 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4612..4629) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4783..5371) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4863..4882) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5542..6402) /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 6165..6184 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6403..6507) /gene="bla" /label=AmpR promoter ORIGIN 1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga 61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct 181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca 241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc 841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta 901 gcgtttaaac ttaagcttgc caccatgaaa acgatcatcg ccctgagcta catcttctgc 961 ctggtattcg ccatgtacac cgatatagag atgaacaggc tgggaaagga tagcctcatg 1021 aacgggaccg agggcccaaa cttctacgtg cctttctcca acaagacggg cgtggtgcgc 1081 agccccttcg aggccccgca gtactacctg gcggagccat ggcagttctc catgctggcc 1141 gcctacatgt tcctgctgat catgcttggc ttccccatca acttcctcac gctgtacgtc 1201 tttagttgta ccaccaagaa gagaaccacg ctcaactaca tcctgctcaa cctggccgtg 1261 gccgacctct tcatggtgtt cgggggcttc accaccaccc tctacacctc tctgcacggg 1321 tacttcgtct ttgggcccac gggctgcaac ctggagggct tctttgccac cttgggcggt 1381 gaaattgcac tgtggtcctt ggtggtcctg gccatcgagc ggtacgtggt ggtggtacag 1441 ccgaagtacg ccaaagaact taaaaacacg tgcaaagcca tcatgggcgt cgccttcacc 1501 tgggtcatgg ctctggcctg tgccgcgccc cccctcgtcg gctggtccag gtacatcccg 1561 gagggcatgc agtgctcgtg cgggattgac tactacacgc cccacgagga aaccaacaat 1621 gagtcgttcg tcatctacat gttcgtggtc cacttcatca tccccctgat tgtcatattc 1681 ttctgctacg gagtcattat tcataatctc cttcacggca ggacgtctaa gctgaaaccc 1741 aaagtcaagg agaagtccat acgcatggtg atcatcatgg tcatcgcttt cctaatctgc 1801 tggctgccct acgctggggt ggcgttctac atcttcaccc atcagggctc tgactttggc 1861 cccatcttca tgaccatccc ggctttcttt gccaagactt ctgccgtcta caaccccgtc 1921 atctacatca tgatgaacaa gcagttccgc gctcgagtca ttagtgtcat gctataccgt 1981 aattaccttc gaagcatgcg cagaaaaagt ttccgatctg gtagtctacg gtcactaagc 2041 aatataaaca gtgaaatgtt aacagaaacc agccaagtgg cgcctgccta actcgagtct 2101 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 2161 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 2221 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 2281 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 2341 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 2401 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 2461 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 2521 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 2581 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 2641 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 2701 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 2761 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 2821 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 2881 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 2941 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 3001 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 3061 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 3121 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 3181 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc 3241 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat 3301 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt 3361 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac 3421 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg 3481 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc 3541 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa 3601 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc 3661 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg 3721 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg 3781 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa 3841 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg 3901 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct 3961 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc 4021 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa 4081 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat 4141 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt 4201 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 4261 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 4321 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg 4381 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 4441 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 4501 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 4561 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 4621 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 4681 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 4741 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 4801 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 4861 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 4921 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 4981 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 5041 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 5101 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 5161 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 5221 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 5281 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 5341 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 5401 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 5461 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 5521 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 5581 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 5641 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 5701 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 5761 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 5821 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 5881 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 5941 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 6001 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 6061 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 6121 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 6181 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 6241 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 6301 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 6361 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 6421 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 6481 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacg //