LOCUS Exported 7663 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION encodes a donor vector for recombination with the Bxb1 recombinase with variant G315T of the Beta-2 Adrenergic Receptor and a cAMP-responsive luciferase reporter gene. ACCESSION . VERSION . KEYWORDS Bxb1Donor_G315T_ADRB2_LucReporter SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7663) TITLE Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning JOURNAL Unpublished REFERENCE 2 (bases 1 to 7663) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7663 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 59..78 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" regulatory 163..172 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 172..237 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" misc_feature 1489..2062 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" misc_feature 1512..2064 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(1656..1673) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 1883..1902 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" polyA_signal 2514..2738 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 3202..3233 /label=minP /note="minimal TATA-box promoter with low basal activity" regulatory 3260..3269 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3266..4918 /codon_start=1 /product="firefly luciferase" /label=luciferase /note="synthetic luc2 version of the luciferase gene" /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV" polyA_signal 5249..5370 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5286..5305) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5340..5359 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5395..5411) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5395..5411) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5408..5430) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5419..5435 /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(5443..5473) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5488..5509 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5626..5643) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5797..6385) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5877..5896) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6556..7371) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 7279..7298 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(7372..7476) /gene="bla" /label=AmpR promoter primer_bind 7544..7562 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(7600..7622) /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 61 gcatcagagc agattgtact gagagtgcac aagcttccgg cttgtcgacg acggcggtct 121 ccgtcgtcag gatcatccgg ggtagtaata agggcgaccg ccgccaccat ggactacaaa 181 gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga tgataaagga 241 tccggccagc ctggcaatgg cagcgccttt ctgctggccc ctaatggctc tcacgccccc 301 gatcacgacg tgacccagga acgggatgaa gtgtgggtcg tgggcatggg catcgtgatg 361 agcctgatcg tgctggccat cgtgttcggc aacgtgctcg tgatcaccgc tatcgccaag 421 ttcgagcggc tgcagaccgt gaccaactac ttcatcacct ccctggcctg cgccgacctc 481 gtgatgggac tggctgtggt gccttttgga gccgcccaca tcctgatgaa gatgtggacc 541 ttcggcaact tttggtgcga gttctggacc agcatcgacg tgctgtgcgt gaccgcctcc 601 atcgagacac tgtgtgtgat cgccgtggac agatacttcg ccatcaccag ccccttcaag 661 taccagagcc tgctgaccaa gaacaaggcc agagtgatca ttctgatggt gtggatcgtg 721 tccggcctga ccagcttcct gcccatccag atgcactggt acagagccac ccaccaggaa 781 gccatcaact gctacgccaa cgaaacctgc tgcgacttct tcacaaacca ggcctacgcc 841 attgcctcca gcatcgtgtc cttctacgtg cccctcgtga ttatggtgtt cgtgtacagc 901 cgggtgttcc aggaagctaa gcggcagctg cagaagatcg acaagagcga gggccggttc 961 cacgtgcaga acctgagcca ggtggaacag gacggcagaa caggccacgg cctgcggaga 1021 agcagcaagt tctgcctgaa agagcacaag gccctgaaaa ccctgggcat catcatgggc 1081 accttcaccc tgtgctggct gcccttcttc atcgtgaaca tcgtgcacgt gatccaggac 1141 aacctgatcc ggaaagaggt gtacatcctg ctgaactgga tcacatacgt gaacagcggc 1201 ttcaaccccc tgatctactg cagaagcccc gacttccgga tcgccttcca ggaactgctg 1261 tgcctgagaa gaagcagcct gaaggcctac ggcaacggct acagcagcaa cggcaatacc 1321 ggcgagcaga gcggctacca tgtggaacag gaaaaagaaa acaagctgct gtgcgaggac 1381 ctgcccggca ccgaggattt tgtgggccac cagggaaccg tgcccagcga caacatcgac 1441 agccagggca gaaactgcag caccaacgac agcctgctgt gagctagccc cctctccctc 1501 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta 1561 tatgtgattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc 1621 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct 1681 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt 1741 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa 1801 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg 1861 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga 1921 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac 1981 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt 2041 cctttgaaaa acacgatgat aatatgaaaa ccttcaacat ctctcagcag gatctggagc 2101 tggtggaggt cgccactgag aagatcacca tgctctatga ggacaacaag caccatgtcg 2161 gggcggccat caggaccaag actggggaga tcatctctgc tgtccacatt gaagcctaca 2221 ttggcagggt cactgtctgt gctgaagcca ttgccattgg gtctgctgtg agcaacgggc 2281 agaaggactt tgacaccatt gtggctgtca ggcaccccta ctctgatgag gtggacagat 2341 ccatcagggt ggtcagcccc tgtggcatgt gtagagagct gatctctgac tatgctcctg 2401 actgctttgt gctcattgag atgaatggca agctggtcaa aaccaccatt gaggaactca 2461 tccccctcaa gtacaccagg aactaatctc gttcgaaaat catcgcggcc cgactgtgcc 2521 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 2581 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 2641 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 2701 caatagcagg catgctgggg atgcggtggg ctctatggca cttttatgag ggacagcccc 2761 ccccccaaag cccccaggga tgtaattacg tccctccccc gctagggggc agcagcgagc 2821 cgcccggggc tccgctccgg tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg 2881 ggacagcccg ggcacgggga aggtggcacg ggatcgcttt cctctgaacg cttctcgctg 2941 ctctttgagc ctgcagacac ctggggggat acggggaaaa ggacgcgtgc accagacagt 3001 gacgtcagct gccagatccc atggccgtca tactgtgacg tctttcagac agcaccagac 3061 agtgacgtca gctgccagat cccatggccg tcatactgtg acgtctttca gacaccccat 3121 tgacgtcaat gggagaacgg tacctgagct cgctagcctc gaggatatca agatctggcc 3181 tcggcggcca agcttagaca ctagagggta tataatggaa gctcgacttc cagcttggca 3241 atccggtact gttggtaaag ccaccatgga agatgccaaa aacattaaga agggcccagc 3301 gccattctac ccactcgaag acgggaccgc cggcgagcag ctgcacaaag ccatgaagcg 3361 ctacgccctg gtgcccggca ccatcgcctt taccgacgca catatcgagg tggacattac 3421 ctacgccgag tacttcgaga tgagcgttcg gctggcagaa gctatgaagc gctatgggct 3481 gaatacaaac catcggatcg tggtgtgcag cgagaatagc ttgcagttct tcatgcccgt 3541 gttgggtgcc ctgttcatcg gtgtggctgt ggccccagct aacgacatct acaacgagcg 3601 cgagctgctg aacagcatgg gcatcagcca gcccaccgtc gtattcgtga gcaagaaagg 3661 gctgcaaaag atcctcaacg tgcaaaagaa gctaccgatc atacaaaaga tcatcatcat 3721 ggatagcaag accgactacc agggcttcca aagcatgtac accttcgtga cttcccattt 3781 gccacccggc ttcaacgagt acgacttcgt gcccgagagc ttcgaccggg acaaaaccat 3841 cgccctgatc atgaacagta gtggcagtac cggattgccc aagggcgtag ccctaccgca 3901 ccgcaccgct tgtgtccgat tcagtcatgc ccgcgacccc atcttcggca accagatcat 3961 ccccgacacc gctatcctca gcgtggtgcc atttcaccac ggcttcggca tgttcaccac 4021 gctgggctac ttgatctgcg gctttcgggt cgtgctcatg taccgcttcg aggaggagct 4081 attcttgcgc agcttgcaag actataagat tcaatctgcc ctgctggtgc ccacactatt 4141 tagcttcttc gctaagagca ctctcatcga caagtacgac ctaagcaact tgcacgagat 4201 cgccagcggc ggggcgccgc tcagcaagga ggtaggtgag gccgtggcca aacgcttcca 4261 cctaccaggc atccgccagg gctacggcct gacagaaaca accagcgcca ttctgatcac 4321 ccccgaaggg gacgacaagc ctggcgcagt aggcaaggtg gtgcccttct tcgaggctaa 4381 ggtggtggac ttggacaccg gcaagacact gggtgtgaac cagcgcggcg agctgtgcgt 4441 ccgtggcccc atgatcatga gcggctacgt taacaacccc gaggctacaa acgctctcat 4501 cgacaaggac ggctggctgc acagcggcga catcgcctac tgggacgagg acgagcactt 4561 cttcatcgtg gaccggctga agagcctgat caaatacaag ggctaccagg tagccccagc 4621 cgaactggag agcatcctgc tgcaacaccc caacatcttc gacgccgggg tcgccggcct 4681 gcccgacgac gatgccggcg agctgcccgc cgcagtcgtc gtgctggaac acggtaaaac 4741 catgaccgag aaggagatcg tggactatgt ggccagccag gttacaaccg ccaagaagct 4801 gcgcggtggt gttgtgttcg tggacgaggt gcctaaagga ctgaccggca agttggacgc 4861 ccgcaagatc cgcgagattc tcattaaggc caagaagggc ggcaagatcg ccgtgtaata 4921 ataccggtgc ttcacaaaca cagcgacgca tctgattaaa gggggtacct tatgttcaca 4981 actggcgtga atgtaactgt attcagcgat gacccacaca ggcatagagt gtctgctatt 5041 aataactatg ctcaaaaatt gtgtaccttt agctttttaa tttgtaaagg ggttaataag 5101 gaatatttga tgtatagtgc cttgactaga atctagataa ctgatcataa tcagccatac 5161 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa 5221 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa 5281 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 5341 tggtttgtcc aaactcatca atgtatctta ggcgcgccgt taacgatatc catggtcata 5401 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 5461 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 5521 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 5581 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 5641 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 5701 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 5761 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 5821 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 5881 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 5941 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 6001 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 6061 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 6121 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 6181 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 6241 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 6301 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 6361 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 6421 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 6481 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 6541 aacttggtct gacagttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat 6601 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc 6661 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc 6721 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc 6781 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac 6841 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt 6901 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt 6961 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc 7021 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccaggga tcgcagtggt 7081 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa 7141 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt 7201 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc 7261 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt 7321 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca tactcttcct 7381 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 7441 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 7501 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 7561 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 7621 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc aga //