LOCUS Exported 7663 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION encodes a donor vector for recombination with the Bxb1 recombinase with variant N51L of the Beta-2 Adrenergic Receptor and a cAMP-responsive luciferase reporter gene. ACCESSION . VERSION . KEYWORDS Bxb1Donor_N51L_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TITLE Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning JOURNAL Unpublished REFERENCE 2 (bases 1 to 7663) AUTHORS .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 71..90 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" regulatory 175..184 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 184..249 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" misc_feature 1501..2074 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" misc_feature 1524..2076 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(1668..1685) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 1895..1914 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" polyA_signal 2526..2750 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 3214..3245 /label=minP /note="minimal TATA-box promoter with low basal activity" regulatory 3272..3281 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3278..4930 /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 5261..5382 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5298..5317) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5352..5371 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5407..5423) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5407..5423) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5420..5442) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5431..5447 /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(5455..5485) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5500..5521 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5638..5655) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5809..6397) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5889..5908) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6568..7383) /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 7291..7310 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(7384..7488) /gene="bla" /label=AmpR promoter primer_bind 7556..7574 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(7612..7634) /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg 61 cttaactatg cggcatcaga gcagattgta ctgagagtgc acaagcttcc ggcttgtcga 121 cgacggcggt ctccgtcgtc aggatcatcc ggggtagtaa taagggcgac cgccgccacc 181 atggactaca aagaccatga cggtgattat aaagatcatg acatcgatta caaggatgac 241 gatgataaag gatccggcca gcctggcaat ggcagcgcct ttctgctggc ccctaatggc 301 tctcacgccc ccgatcacga cgtgacccag gaacgggatg aagtgtgggt cgtgggcatg 361 ggcatcgtga tgagcctgat cgtgctggcc atcgtgttcg gcctcgtgct cgtgatcacc 421 gctatcgcca agttcgagcg gctgcagacc gtgaccaact acttcatcac ctccctggcc 481 tgcgccgacc tcgtgatggg actggctgtg gtgccttttg gagccgccca catcctgatg 541 aagatgtgga ccttcggcaa cttttggtgc gagttctgga ccagcatcga cgtgctgtgc 601 gtgaccgcct ccatcgagac actgtgtgtg atcgccgtgg acagatactt cgccatcacc 661 agccccttca agtaccagag cctgctgacc aagaacaagg ccagagtgat cattctgatg 721 gtgtggatcg tgtccggcct gaccagcttc ctgcccatcc agatgcactg gtacagagcc 781 acccaccagg aagccatcaa ctgctacgcc aacgaaacct gctgcgactt cttcacaaac 841 caggcctacg ccattgcctc cagcatcgtg tccttctacg tgcccctcgt gattatggtg 901 ttcgtgtaca gccgggtgtt ccaggaagct aagcggcagc tgcagaagat cgacaagagc 961 gagggccggt tccacgtgca gaacctgagc caggtggaac aggacggcag aacaggccac 1021 ggcctgcgga gaagcagcaa gttctgcctg aaagagcaca aggccctgaa aaccctgggc 1081 atcatcatgg gcaccttcac cctgtgctgg ctgcccttct tcatcgtgaa catcgtgcac 1141 gtgatccagg acaacctgat ccggaaagag gtgtacatcc tgctgaactg gatcggctac 1201 gtgaacagcg gcttcaaccc cctgatctac tgcagaagcc ccgacttccg gatcgccttc 1261 caggaactgc tgtgcctgag aagaagcagc ctgaaggcct acggcaacgg ctacagcagc 1321 aacggcaata ccggcgagca gagcggctac catgtggaac aggaaaaaga aaacaagctg 1381 ctgtgcgagg acctgcccgg caccgaggat tttgtgggcc accagggaac cgtgcccagc 1441 gacaacatcg acagccaggg cagaaactgc agcaccaacg acagcctgct gtgagctagc 1501 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 1561 tgcgtttgtc tatatgtgat tttccaccat attgccgtct tttggcaatg tgagggcccg 1621 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 1681 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 1741 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 1801 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 1861 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 1921 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 1981 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 2041 ggacgtggtt ttcctttgaa aaacacgatg ataatatgaa aaccttcaac atctctcagc 2101 aggatctgga gctggtggag gtcgccactg agaagatcac catgctctat gaggacaaca 2161 agcaccatgt cggggcggcc atcaggacca agactgggga gatcatctct gctgtccaca 2221 ttgaagccta cattggcagg gtcactgtct gtgctgaagc cattgccatt gggtctgctg 2281 tgagcaacgg gcagaaggac tttgacacca ttgtggctgt caggcacccc tactctgatg 2341 aggtggacag atccatcagg gtggtcagcc cctgtggcat gtgtagagag ctgatctctg 2401 actatgctcc tgactgcttt gtgctcattg agatgaatgg caagctggtc aaaaccacca 2461 ttgaggaact catccccctc aagtacacca ggaactaatc tcgttcgaaa atcatcgcgg 2521 cccgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 2581 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 2641 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 2701 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cacttttatg 2761 agggacagcc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg 2821 gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc 2881 ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa 2941 cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa aaggacgcgt 3001 gcaccagaca gtgacgtcag ctgccagatc ccatggccgt catactgtga cgtctttcag 3061 acagcaccag acagtgacgt cagctgccag atcccatggc cgtcatactg tgacgtcttt 3121 cagacacccc attgacgtca atgggagaac ggtacctgag ctcgctagcc tcgaggatat 3181 caagatctgg cctcggcggc caagcttaga cactagaggg tatataatgg aagctcgact 3241 tccagcttgg caatccggta ctgttggtaa agccaccatg gaagatgcca aaaacattaa 3301 gaagggccca gcgccattct acccactcga agacgggacc gccggcgagc agctgcacaa 3361 agccatgaag cgctacgccc tggtgcccgg caccatcgcc tttaccgacg cacatatcga 3421 ggtggacatt acctacgccg agtacttcga gatgagcgtt cggctggcag aagctatgaa 3481 gcgctatggg ctgaatacaa accatcggat cgtggtgtgc agcgagaata gcttgcagtt 3541 cttcatgccc gtgttgggtg ccctgttcat cggtgtggct gtggccccag ctaacgacat 3601 ctacaacgag cgcgagctgc tgaacagcat gggcatcagc cagcccaccg tcgtattcgt 3661 gagcaagaaa gggctgcaaa agatcctcaa cgtgcaaaag aagctaccga tcatacaaaa 3721 gatcatcatc atggatagca agaccgacta ccagggcttc caaagcatgt acaccttcgt 3781 gacttcccat ttgccacccg gcttcaacga gtacgacttc gtgcccgaga gcttcgaccg 3841 ggacaaaacc atcgccctga tcatgaacag tagtggcagt accggattgc ccaagggcgt 3901 agccctaccg caccgcaccg cttgtgtccg attcagtcat gcccgcgacc ccatcttcgg 3961 caaccagatc atccccgaca ccgctatcct cagcgtggtg ccatttcacc acggcttcgg 4021 catgttcacc acgctgggct acttgatctg cggctttcgg gtcgtgctca tgtaccgctt 4081 cgaggaggag ctattcttgc gcagcttgca agactataag attcaatctg ccctgctggt 4141 gcccacacta tttagcttct tcgctaagag cactctcatc gacaagtacg acctaagcaa 4201 cttgcacgag atcgccagcg gcggggcgcc gctcagcaag gaggtaggtg aggccgtggc 4261 caaacgcttc cacctaccag gcatccgcca gggctacggc ctgacagaaa caaccagcgc 4321 cattctgatc acccccgaag gggacgacaa gcctggcgca gtaggcaagg tggtgccctt 4381 cttcgaggct aaggtggtgg acttggacac cggcaagaca ctgggtgtga accagcgcgg 4441 cgagctgtgc gtccgtggcc ccatgatcat gagcggctac gttaacaacc ccgaggctac 4501 aaacgctctc atcgacaagg acggctggct gcacagcggc gacatcgcct actgggacga 4561 ggacgagcac ttcttcatcg tggaccggct gaagagcctg atcaaataca agggctacca 4621 ggtagcccca gccgaactgg agagcatcct gctgcaacac cccaacatct tcgacgccgg 4681 ggtcgccggc ctgcccgacg acgatgccgg cgagctgccc gccgcagtcg tcgtgctgga 4741 acacggtaaa accatgaccg agaaggagat cgtggactat gtggccagcc aggttacaac 4801 cgccaagaag ctgcgcggtg gtgttgtgtt cgtggacgag gtgcctaaag gactgaccgg 4861 caagttggac gcccgcaaga tccgcgagat tctcattaag gccaagaagg gcggcaagat 4921 cgccgtgtaa taataccggt caccctccgt accaacgacg catctgatta aagggggtac 4981 cttatgttca caactggcgt gaatgtaact gtattcagcg atgacccaca caggcataga 5041 gtgtctgcta ttaataacta tgctcaaaaa ttgtgtacct ttagcttttt aatttgtaaa 5101 ggggttaata aggaatattt gatgtatagt gccttgacta gaatctagat aactgatcat 5161 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc 5221 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta 5281 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 5341 gcattctagt tgtggtttgt ccaaactcat caatgtatct taggcgcgcc gttaacgata 5401 tccatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 5461 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 5521 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 5581 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 5641 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 5701 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 5761 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 5821 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 5881 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 5941 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 6001 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 6061 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 6121 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 6181 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 6241 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 6301 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 6361 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 6421 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 6481 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 6541 tatatatgag taaacttggt ctgacagtta gaaaaactca tcgagcatca aatgaaactg 6601 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga 6661 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat 6721 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc 6781 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat 6841 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc 6901 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt 6961 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc 7021 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccagg 7081 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg 7141 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc 7201 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg 7261 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc 7321 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct 7381 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 7441 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 7501 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 7561 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca 7621 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gat //