LOCUS Exported 6610 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expression of a chimeric G-protein coupled receptor (Rhodopsin-GPR34; Opto-GPR34; B7). ACCESSION . VERSION . KEYWORDS Opto-GPR34 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6610) 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 6610) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6610 /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(2040..2057) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2046..2270 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2316..2744 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2403..2422) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2613..2634 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2753..2773) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2758..3087 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2938..3073 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3000..3019 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3154..3948 /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(3208..3227) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3818..3837 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4122..4243 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4159..4178) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4213..4232 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4292..4308) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4292..4308) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4305..4327) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4316..4332 /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(4340..4370) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4385..4406 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4523..4540) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4694..5282) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4774..4793) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5453..6313) /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 6076..6095 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6314..6418) /gene="bla" /label=AmpR promoter primer_bind complement(6493..6512) /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 gggtattcac cgtaaaagaa attccctcaa 1081 ctacatcctg ctcaacctgg ccgtggccga cctcttcatg gtgttcgggg gcttcaccac 1141 caccctctac acctctctgc acgggtactt cgtctttggg cccacgggct gcaacctgga 1201 gggcttcttt gccaccttgg gcggtgaaat tgcactgtgg tccttggtgg tcctggccat 1261 cgagcggtac gtggtggtga atcggtctat acagcaacgg aaggcaataa caaccaaaca 1321 agccatcatg ggcgtcgcct tcacctgggt catggctctg gcctgtgccg cgccccccct 1381 cgtcggctgg tccaggtaca tcccggaggg catgcagtgc tcgtgcggga ttgactacta 1441 cacgccccac gaggaaacca acaatgagtc gttcgtcatc tacatgttcg tggtccactt 1501 catcatcccc ctgattgtca tattcttctg ctacggaaag attgggaaga atctattgag 1561 gatttctaaa aggaggtcaa aatttcctaa ttctggtaaa tatgccacta cagctcgcat 1621 ggtgatcatc atggtcatcg ctttcctaat ctgctggctg ccctacgctg gggtggcgtt 1681 ctacatcttc acccatcagg gctctgactt tggccccatc ttcatgacca tcccggcttt 1741 ctttgccaag acttctgccg tctacaaccc cgtcatctac atcatgatga acaagcagtt 1801 ccgcaaaata atgtgccaac ttctttttag acgatttcaa ggtgaaccaa gtaggagtga 1861 aagcacttca gaatttaaac caggatactc cctgcatgat acatctgtgg cagtgaaaat 1921 acagtctagt tctaaaagta ctacagaaac cagccaagtg gcgcctgcct aagaattcca 1981 ccacactgga ctagtggatc cgagctcggt accaagctta agtttaaacc gctgatcagc 2041 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 2101 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 2161 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 2221 ggattgggaa gagaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc 2281 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag 2341 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 2401 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 2461 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 2521 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 2581 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 2641 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 2701 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg 2761 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 2821 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt 2881 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc 2941 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt 3001 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc 3061 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga 3121 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca 3181 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc 3241 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc 3301 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg 3361 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg 3421 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct 3481 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct 3541 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa 3601 gccggtcttg tcgatcagga tgatctggac gaagaacatc aggggctcgc gccagccgaa 3661 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc 3721 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt 3781 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct 3841 gaagaacttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc 3901 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg 3961 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg 4021 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc 4081 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt 4141 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 4201 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt 4261 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 4321 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 4381 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 4441 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 4501 gtattgggcg ctcttgcgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 4561 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 4621 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 4681 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 4741 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 4801 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 4861 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 4921 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 4981 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 5041 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 5101 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 5161 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 5221 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 5281 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 5341 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 5401 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 5461 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 5521 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 5581 caatgatacc gcgggaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 5641 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 5701 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 5761 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 5821 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 5881 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 5941 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 6001 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 6061 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 6121 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc actgttgaga tccagttcga 6181 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 6241 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 6301 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 6361 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 6421 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg 6481 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc 6541 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg 6601 cttgaccgac //