LOCUS       sequence-255165-        7663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes a donor vector for recombination with the Bxb1 recombinase 
            with variant W99A of the Beta-2 Adrenergic Receptor and a 
            cAMP-responsive luciferase reporter gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7663)
  AUTHORS   Jones EM, Lubock NB, Venkatakrishnan AJ, Wang J, Tseng AM, Paggi JM,
            Latorraca NR, Cancilla D, Satyadi M, Davis JE, Babu MM, Dror RO, 
            Kosuri S
  TITLE     Structural and functional characterization of G protein-coupled 
            receptors with deep mutational scanning.
  JOURNAL   Elife. 2020 Oct 21;9. pii: 54895. doi: 10.7554/eLife.54895.
  PUBMED    33084570
REFERENCE   2  (bases 1 to 7663)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2020-10-21"; volume: "9. pii: 54895. doi"; pages: " 
            10.7554/eLife.54895"
COMMENT     Sequence Label: Bxb1Donor_W99A_ADRB2_LucReporter
FEATURES             Location/Qualifiers
     source          1..7663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     111..130
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    151..188
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      215..224
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             224..289
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    1541..2114
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1564..2116
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1708..1725)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1935..1954
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    2566..2790
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3254..3285
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      3312..3321
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3318..4970
                     /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    5301..5422
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5338..5357)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5392..5411
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5447..5463)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5447..5463)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5460..5482)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5471..5487
                     /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(join(5495..5501,5502..5519,5520..5525))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5540..5561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5678..5695)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5849..6437)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5929..5948)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6608..7423)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6608..7423)
                     /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     7331..7350
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(7424..7528)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7596..7614
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(join(7652..7663,1..11))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
       61 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      121 gcagattgta ctgagagtgc acaagcttcc ggcttgtcga cgacggcggt ctccgtcgtc
      181 aggatcatcc ggggtagtaa taagggcgac cgccgccacc atggactaca aagaccatga
      241 cggtgattat aaagatcatg acatcgatta caaggatgac gatgataaag gatccggcca
      301 gcctggcaat ggcagcgcct ttctgctggc ccctaatggc tctcacgccc ccgatcacga
      361 cgtgacccag gaacgggatg aagtgtgggt cgtgggcatg ggcatcgtga tgagcctgat
      421 cgtgctggcc atcgtgttcg gcaacgtgct cgtgatcacc gctatcgcca agttcgagcg
      481 gctgcagacc gtgaccaact acttcatcac ctccctggcc tgcgccgacc tcgtgatggg
      541 actggctgtg gtgccttttg gagccgccca catcctgatg aagatggcta ccttcggcaa
      601 cttttggtgc gagttctgga ccagcatcga cgtgctgtgc gtgaccgcct ccatcgagac
      661 actgtgtgtg atcgccgtgg acagatactt cgccatcacc agccccttca agtaccagag
      721 cctgctgacc aagaacaagg ccagagtgat cattctgatg gtgtggatcg tgtccggcct
      781 gaccagcttc ctgcccatcc agatgcactg gtacagagcc acccaccagg aagccatcaa
      841 ctgctacgcc aacgaaacct gctgcgactt cttcacaaac caggcctacg ccattgcctc
      901 cagcatcgtg tccttctacg tgcccctcgt gattatggtg ttcgtgtaca gccgggtgtt
      961 ccaggaagct aagcggcagc tgcagaagat cgacaagagc gagggccggt tccacgtgca
     1021 gaacctgagc caggtggaac aggacggcag aacaggccac ggcctgcgga gaagcagcaa
     1081 gttctgcctg aaagagcaca aggccctgaa aaccctgggc atcatcatgg gcaccttcac
     1141 cctgtgctgg ctgcccttct tcatcgtgaa catcgtgcac gtgatccagg acaacctgat
     1201 ccggaaagag gtgtacatcc tgctgaactg gatcggctac gtgaacagcg gcttcaaccc
     1261 cctgatctac tgcagaagcc ccgacttccg gatcgccttc caggaactgc tgtgcctgag
     1321 aagaagcagc ctgaaggcct acggcaacgg ctacagcagc aacggcaata ccggcgagca
     1381 gagcggctac catgtggaac aggaaaaaga aaacaagctg ctgtgcgagg acctgcccgg
     1441 caccgaggat tttgtgggcc accagggaac cgtgcccagc gacaacatcg acagccaggg
     1501 cagaaactgc agcaccaacg acagcctgct gtgagctagc cccctctccc tccccccccc
     1561 ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgtgat
     1621 tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct
     1681 tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg
     1741 tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc
     1801 tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg
     1861 tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg
     1921 tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga
     1981 aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt
     2041 agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa
     2101 aaacacgatg ataatatgaa aaccttcaac atctctcagc aggatctgga gctggtggag
     2161 gtcgccactg agaagatcac catgctctat gaggacaaca agcaccatgt cggggcggcc
     2221 atcaggacca agactgggga gatcatctct gctgtccaca ttgaagccta cattggcagg
     2281 gtcactgtct gtgctgaagc cattgccatt gggtctgctg tgagcaacgg gcagaaggac
     2341 tttgacacca ttgtggctgt caggcacccc tactctgatg aggtggacag atccatcagg
     2401 gtggtcagcc cctgtggcat gtgtagagag ctgatctctg actatgctcc tgactgcttt
     2461 gtgctcattg agatgaatgg caagctggtc aaaaccacca ttgaggaact catccccctc
     2521 aagtacacca ggaactaatc tcgttcgaaa atcatcgcgg cccgactgtg ccttctagtt
     2581 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2641 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2701 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2761 ggcatgctgg ggatgcggtg ggctctatgg cacttttatg agggacagcc ccccccccaa
     2821 agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg
     2881 gctccgctcc ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc
     2941 cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga
     3001 gcctgcagac acctgggggg atacggggaa aaggacgcgt gcaccagaca gtgacgtcag
     3061 ctgccagatc ccatggccgt catactgtga cgtctttcag acagcaccag acagtgacgt
     3121 cagctgccag atcccatggc cgtcatactg tgacgtcttt cagacacccc attgacgtca
     3181 atgggagaac ggtacctgag ctcgctagcc tcgaggatat caagatctgg cctcggcggc
     3241 caagcttaga cactagaggg tatataatgg aagctcgact tccagcttgg caatccggta
     3301 ctgttggtaa agccaccatg gaagatgcca aaaacattaa gaagggccca gcgccattct
     3361 acccactcga agacgggacc gccggcgagc agctgcacaa agccatgaag cgctacgccc
     3421 tggtgcccgg caccatcgcc tttaccgacg cacatatcga ggtggacatt acctacgccg
     3481 agtacttcga gatgagcgtt cggctggcag aagctatgaa gcgctatggg ctgaatacaa
     3541 accatcggat cgtggtgtgc agcgagaata gcttgcagtt cttcatgccc gtgttgggtg
     3601 ccctgttcat cggtgtggct gtggccccag ctaacgacat ctacaacgag cgcgagctgc
     3661 tgaacagcat gggcatcagc cagcccaccg tcgtattcgt gagcaagaaa gggctgcaaa
     3721 agatcctcaa cgtgcaaaag aagctaccga tcatacaaaa gatcatcatc atggatagca
     3781 agaccgacta ccagggcttc caaagcatgt acaccttcgt gacttcccat ttgccacccg
     3841 gcttcaacga gtacgacttc gtgcccgaga gcttcgaccg ggacaaaacc atcgccctga
     3901 tcatgaacag tagtggcagt accggattgc ccaagggcgt agccctaccg caccgcaccg
     3961 cttgtgtccg attcagtcat gcccgcgacc ccatcttcgg caaccagatc atccccgaca
     4021 ccgctatcct cagcgtggtg ccatttcacc acggcttcgg catgttcacc acgctgggct
     4081 acttgatctg cggctttcgg gtcgtgctca tgtaccgctt cgaggaggag ctattcttgc
     4141 gcagcttgca agactataag attcaatctg ccctgctggt gcccacacta tttagcttct
     4201 tcgctaagag cactctcatc gacaagtacg acctaagcaa cttgcacgag atcgccagcg
     4261 gcggggcgcc gctcagcaag gaggtaggtg aggccgtggc caaacgcttc cacctaccag
     4321 gcatccgcca gggctacggc ctgacagaaa caaccagcgc cattctgatc acccccgaag
     4381 gggacgacaa gcctggcgca gtaggcaagg tggtgccctt cttcgaggct aaggtggtgg
     4441 acttggacac cggcaagaca ctgggtgtga accagcgcgg cgagctgtgc gtccgtggcc
     4501 ccatgatcat gagcggctac gttaacaacc ccgaggctac aaacgctctc atcgacaagg
     4561 acggctggct gcacagcggc gacatcgcct actgggacga ggacgagcac ttcttcatcg
     4621 tggaccggct gaagagcctg atcaaataca agggctacca ggtagcccca gccgaactgg
     4681 agagcatcct gctgcaacac cccaacatct tcgacgccgg ggtcgccggc ctgcccgacg
     4741 acgatgccgg cgagctgccc gccgcagtcg tcgtgctgga acacggtaaa accatgaccg
     4801 agaaggagat cgtggactat gtggccagcc aggttacaac cgccaagaag ctgcgcggtg
     4861 gtgttgtgtt cgtggacgag gtgcctaaag gactgaccgg caagttggac gcccgcaaga
     4921 tccgcgagat tctcattaag gccaagaagg gcggcaagat cgccgtgtaa taataccggt
     4981 taaaagacca aatcacgacg catctgatta aagggggtac cttatgttca caactggcgt
     5041 gaatgtaact gtattcagcg atgacccaca caggcataga gtgtctgcta ttaataacta
     5101 tgctcaaaaa ttgtgtacct ttagcttttt aatttgtaaa ggggttaata aggaatattt
     5161 gatgtatagt gccttgacta gaatctagat aactgatcat aatcagccat accacatttg
     5221 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     5281 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     5341 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5401 ccaaactcat caatgtatct taggcgcgcc gttaacgata tccatggtca tagctgtttc
     5461 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5521 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5581 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5641 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5701 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5761 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5821 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5881 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5941 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6001 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6061 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6121 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6181 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6241 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6301 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6361 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6421 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6481 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6541 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6601 ctgacagtta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     6661 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     6721 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     6781 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     6841 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     6901 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     6961 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     7021 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     7081 caggatattc ttctaatacc tggaatgctg ttttcccagg gatcgcagtg gtgagtaacc
     7141 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     7201 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     7261 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     7321 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     7381 atcgcggcct agagcaagac gtttcccgtt gaatatggct catactcttc ctttttcaat
     7441 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7501 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     7561 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     7621 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca cat
//
