LOCUS Exported 7751 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of G protein-coupled receptors for PRESTO-Tango: parallel receptorome expression and screening via transcriptional output, with transcriptional activation following arrestin translocation. ACCESSION . VERSION . KEYWORDS CMKLR1-Tango SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7751) AUTHORS Kroeze WK, Sassano MF, Huang XP, Lansu K, McCorvy JD, Giguere PM, Sciaky N, Roth BL TITLE PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. JOURNAL Nat Struct Mol Biol. 2015 May;22(5):362-9. doi: 10.1038/nsmb.3014. Epub 2015 Apr 20. PUBMED 25895059 REFERENCE 2 (bases 1 to 7751) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7751 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 2306..3307 /codon_start=1 /product="tetracycline-controlled transactivator, comprising a fusion of the tetracycline repressor TetR with the C-terminal activation domain of herpes simplex virus VP16" /label=tTA /note="In the Tet-Off(R) system, tTA binds to the Tet-responsive element and stimulates transcription only in the absence of tetracycline or doxycycline." /translation="RLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKN KRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPT EKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDS MPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEG LLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADA LDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG" primer_bind complement(2373..2392) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" primer_bind complement(3345..3362) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3351..3575 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3621..4049 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3708..3727) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3918..3939 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4058..4078) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4063..4392 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4243..4378 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4305..4324 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4459..5253 /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(4513..4532) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5123..5142 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5427..5548 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5464..5483) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5518..5537 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5597..5613) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5597..5613) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5610..5632) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5621..5637 /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(5645..5675) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5690..5711 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5828..5845) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5999..6584) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6079..6098) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6755..7615) /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 7378..7397 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7616..7720) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc 961 agatatccag cacagtggcg gccgcgccac catgaagacg atcatcgccc tgagctacat 1021 cttctgcctg gtattcgccg actacaagga cgatgatgac gccagcatcg atatggaaga 1081 cgaagactac aacaccagca tctcttatgg ggacgaatat cccgactacc tggattctat 1141 cgtggtgctg gaagatctga gccctctgga ggcacgcgta accagaattt ttctggtagt 1201 ggtctatagc atcgtttgtt tcctgggcat cctgggcaac ggacttgtta tcataatagc 1261 cacattcaag atgaagaaga ctgtcaacat ggtgtggttt ctgaacctgg ctgtggcgga 1321 cttcctcttc aatgtgtttc tgcctatcca cattacctat gccgcaatgg actaccattg 1381 ggtctttggt accgccatgt gcaaaatctc caatttcctc ttgatacata acatgtttac 1441 ctctgttttt ctcctgacca ttatctcctc cgatcggtgc atttctgtgc tgcttccagt 1501 gtggtctcag aatcacagaa gcgttaggct ggcttatatg gcatgtatgg tgatctgggt 1561 gctggcattt tttctgagca gccctagcct ggtctttcgg gacacagcga atctccacgg 1621 taagatttcc tgcttcaaca acttcagctt gtccaccccg ggcagcagtt cttggccaac 1681 acacagccag atggatcccg taggctattc ccggcacatg gtagtgaccg tgacgcggtt 1741 cctctgcggg ttcctggtgc cagttctcat tataacggca tgttacctga ccatcgtctg 1801 taagctgcaa aggaatcggc ttgctaagac taagaagcca ttcaagatca tcgtcacgat 1861 tatcattacg ttctttctgt gttggtgtcc atatcatact ttgaatcttc tggagctgca 1921 ccacacggcc atgcccggct ctgtcttcag cctgggactc ccattggcca ccgctctcgc 1981 gatcgctaat agttgcatga atcctatttt gtacgtgttt atgggacagg actttaaaaa 2041 gttcaaggtg gccctgttct ctcgcctcgt taacgcactg tccgaggata caggtcattc 2101 cagttatcct agccatcgct cttttaccaa gatgtcttca atgaacgagc gcacaagcat 2161 gaatgaacgc gagaccggca tgcttatcga taccggtgga cgcaccccac ccagcctggg 2221 tccccaagat gagtcctgca ccaccgccag ctcctccctg gccaaggaca cttcatcgac 2281 cggtgagaac ctgtacttcc agctaagatt agataaaagt aaagtgatta acagcgcatt 2341 agagctgctt aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct 2401 aggtgtagag cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc 2461 cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag 2521 ctggcaagat tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg 2581 cgatggagca aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga 2641 aaatcaatta gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact 2701 cagcgctgtg gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc 2761 taaagaagaa agggaaacac ctactactga tagtatgccg ccattattac gacaagctat 2821 cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat 2881 atgcggatta gaaaaacaac ttaaatgtga aagtgggtcc gcgtacagcc gcgcgcgtac 2941 gaaaaacaat tacgggtcta ccatcgaggg cctgctcgat ctcccggacg acgacgcccc 3001 cgaagaggcg gggctggcgg ctccgcgcct gtcctttctc cccgcgggac acacgcgcag 3061 actgtcgacg gcccccccga ccgatgtcag cctgggggac gagctccact tagacggcga 3121 ggacgtggcg atggcgcatg ccgacgcgct agacgatttc gatctggaca tgttggggga 3181 cggggattcc ccgggtccgg gatttacccc ccacgactcc gccccctacg gcgctctgga 3241 tatggccgac ttcgagtttg agcagatgtt taccgatgcc cttggaattg acgagtacgg 3301 tgggtagctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc 3361 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 3421 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 3481 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 3541 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg 3601 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 3661 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 3721 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 3781 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 3841 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 3901 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 3961 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 4021 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt 4081 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 4141 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 4201 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 4261 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 4321 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 4381 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca 4441 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct 4501 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc 4561 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc 4621 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc 4681 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg 4741 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc 4801 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac 4861 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat 4921 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc 4981 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg 5041 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg 5101 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc 5161 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc 5221 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg 5281 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa 5341 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 5401 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 5461 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 5521 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 5581 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 5641 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 5701 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 5761 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 5821 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 5881 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 5941 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 6001 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 6061 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 6121 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 6181 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 6241 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 6301 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 6361 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 6421 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 6481 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggttttt 6541 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 6601 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 6661 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 6721 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 6781 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 6841 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 6901 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 6961 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 7021 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 7081 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 7141 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 7201 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 7261 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 7321 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 7381 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 7441 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 7501 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 7561 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 7621 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 7681 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 7741 cacctgacgt c //