LOCUS Exported 6550 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of a chimeric G-protein coupled receptor (Rhodopsin-GPR31; Opto-GPR31; B4). ACCESSION . VERSION . KEYWORDS Opto-GPR31 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6550) 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 6550) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6550 /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 821..853 /codon_start=1 /product="epitope tag from vesicular stomatitis virus G protein" /label=VSV-G tag /translation="YTDIEMNRLGK" CDS 878..916 /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(1980..1997) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1986..2210 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2256..2684 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2343..2362) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2553..2574 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2693..2713) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2698..3027 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2878..3013 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2940..2959 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3094..3888 /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(3148..3167) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3758..3777 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4062..4183 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4099..4118) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4153..4172 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4232..4248) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4232..4248) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4245..4267) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4256..4272 /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(4280..4310) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4325..4346 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4463..4480) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4634..5222) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4714..4733) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5393..6253) /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 6016..6035 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6254..6358) /gene="bla" /label=AmpR promoter primer_bind complement(6433..6452) /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 gagacacaag ctggctagcg tttaaacggg ccctctagac tcgagcacca tgaaaacgat 781 catcgccctg agctacatct tctgcctggt attcgccatg tacaccgata tagagatgaa 841 caggctggga aaggatagcc tcatgaacgg gaccgagggc ccaaacttct acgtgccttt 901 ctccaacaag acgggcgtgg tgcgcagccc cttcgaggcc ccgcagtact acctggcgga 961 gccatggcag ttctccatgc tggccgccta catgttcctg ctgatcatgc ttggcttccc 1021 catcaacttc ctcacgctgt acgtctttct gttccgggtc agggtgtgga agccgctcaa 1081 ctacatcctg ctcaacctgg ccgtggccga cctcttcatg gtgttcgggg gcttcaccac 1141 caccctctac acctctctgc acgggtactt cgtctttggg cccacgggct gcaacctgga 1201 gggcttcttt gccaccttgg gcggtgaaat tgcactgtgg tccttggtgg tcctggccat 1261 cgagcggtac gtggtggtgg tccaccctcg gcttaaggtc aacctgctgt ctcctcaggc 1321 ggccatcatg ggcgtcgcct tcacctgggt catggctctg gcctgtgccg cgccccccct 1381 cgtcggctgg tccaggtaca tcccggaggg catgcagtgc tcgtgcggga ttgactacta 1441 cacgccccac gaggaaacca acaatgagtc gttcgtcatc tacatgttcg tggtccactt 1501 catcatcccc ctgattgtca tattcttctg ctacggaggc atcatcaggg ctctccagaa 1561 aagactccgg gagcctgaga aacagcccaa gcttcagcgg gcccagcgca tggtgatcat 1621 catggtcatc gctttcctaa tctgctggct gccctacgct ggggtggcgt tctacatctt 1681 cacccatcag ggctctgact ttggccccat cttcatgacc atcccggctt tctttgccaa 1741 gacttctgcc gtctacaacc ccgtcatcta catcatgatg aacaagcagt tccgcagctc 1801 ctatcggagg gtgttccaca ccctccgagg caaagggcag gcagcagagc ccccagattt 1861 caaccccaga gactcctatt ccacagaaac cagccaagtg gcgcctgcct aagaattcca 1921 ccacactgga ctagtggatc cgagctcggt accaagctta agtttaaacc gctgatcagc 1981 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 2041 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 2101 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 2161 ggattgggaa gagaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc 2221 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag 2281 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 2341 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 2401 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 2461 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 2521 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 2581 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 2641 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg 2701 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 2761 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt 2821 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc 2881 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt 2941 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc 3001 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga 3061 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca 3121 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc 3181 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc 3241 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg 3301 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg 3361 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct 3421 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct 3481 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa 3541 gccggtcttg tcgatcagga tgatctggac gaagaacatc aggggctcgc gccagccgaa 3601 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc 3661 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt 3721 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct 3781 gaagaacttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc 3841 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg 3901 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg 3961 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc 4021 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt 4081 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 4141 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt 4201 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 4261 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 4321 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 4381 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 4441 gtattgggcg ctcttgcgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 4501 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 4561 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 4621 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 4681 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 4741 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 4801 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 4861 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 4921 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 4981 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 5041 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 5101 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 5161 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 5221 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 5281 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 5341 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 5401 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 5461 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 5521 caatgatacc gcgggaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 5581 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 5641 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 5701 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 5761 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 5821 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 5881 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 5941 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 6001 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 6061 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc actgttgaga tccagttcga 6121 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 6181 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 6241 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 6301 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 6361 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg 6421 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc 6481 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg 6541 cttgaccgac //