LOCUS Exported 6640 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expression of a chimeric G-protein coupled receptor (Rhodopsin-GPR42; Opto-GPR42; B12). ACCESSION . VERSION . KEYWORDS Opto-GPR42 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6640) 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 6640) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6640 /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(2070..2087) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2076..2300 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2346..2774 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2433..2452) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2643..2664 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2783..2803) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2788..3117 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2968..3103 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3030..3049 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3184..3978 /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(3238..3257) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3848..3867 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4152..4273 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4189..4208) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4243..4262 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4322..4338) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4322..4338) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4335..4357) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4346..4362 /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(4370..4400) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4415..4436 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4553..4570) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4724..5312) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4804..4823) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5483..6343) /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 6106..6125 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6344..6448) /gene="bla" /label=AmpR promoter primer_bind complement(6523..6542) /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 acgtctttgt gggcaagctg cggtgccgcc cggtggccct 1081 caactacatc ctgctcaacc tggccgtggc cgacctcttc atggtgttcg ggggcttcac 1141 caccaccctc tacacctctc tgcacgggta cttcgtcttt gggcccacgg gctgcaacct 1201 ggagggcttc tttgccacct tgggcggtga aattgcactg tggtccttgg tggtcctggc 1261 catcgagcgg tacgtggtgg tggcccaccc actgtggtac aagacccggc cgaggctggg 1321 gcaggccatc atgggcgtcg ccttcacctg ggtcatggct ctggcctgtg ccgcgccccc 1381 cctcgtcggc tggtccaggt acatcccgga gggcatgcag tgctcgtgcg ggattgacta 1441 ctacacgccc cacgaggaaa ccaacaatga gtcgttcgtc atctacatgt tcgtggtcca 1501 cttcatcatc cccctgattg tcatattctt ctgctacgga cgcctggtgt ggatcctcgg 1561 cagagggggc agccaccgcc ggcagaggag ggtggcgcgc atggtgatca tcatggtcat 1621 cgctttccta atctgctggc tgccctacgc tggggtggcg ttctacatct tcacccatca 1681 gggctctgac tttggcccca tcttcatgac catcccggct ttctttgcca agacttctgc 1741 cgtctacaac cccgtcatct acatcatgat gaacaagcag ttccgcgccg actttcatga 1801 gctgctgagg aggttgtgtg ggctctgggg ccagtggcag caggagagca gcatggagct 1861 gaaggagcag aagggagggg aggagcagag agcggaccga ccagctgaaa gaaagaccag 1921 tgaacactca cagggctgtg gaactggtgg ccaggtggcc tgtgctgaaa acacagaaac 1981 cagccaagtg gcgcctgcct aagaattcca ccacactgga ctagtggatc cgagctcggt 2041 accaagctta agtttaaacc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 2101 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 2161 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 2221 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg 2281 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta 2341 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 2401 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 2461 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 2521 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 2581 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 2641 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 2701 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 2761 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc 2821 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga 2881 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 2941 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat 3001 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc 3061 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag 3121 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt 3181 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta 3241 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg 3301 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa 3361 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct 3421 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg 3481 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca 3541 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat 3601 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac 3661 gaagaacatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc 3721 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa 3781 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag 3841 gacatagcgt tggctacccg tgatattgct gaagaacttg gcggcgaatg ggctgaccgc 3901 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt 3961 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca 4021 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa 4081 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct 4141 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 4201 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 4261 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat 4321 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 4381 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 4441 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 4501 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttgcgct tcctcgctca 4561 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4621 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4681 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4741 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4801 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4861 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4921 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4981 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5041 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5101 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5161 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5221 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 5281 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5341 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 5401 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 5461 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 5521 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 5581 gggagggctt accatctggc cccagtgctg caatgatacc gcgggaccca cgctcaccgg 5641 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 5701 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 5761 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 5821 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 5881 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 5941 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 6001 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 6061 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 6121 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 6181 ggatcttacc actgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 6241 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 6301 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 6361 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 6421 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg 6481 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc 6541 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc 6601 gagcaaaatt taagctacaa caaggcaagg cttgaccgac //