LOCUS       sequence-255135-        7663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes a donor vector for recombination with the Bxb1 recombinase 
            with the WT 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_WT_ADRB2_LucReporter
FEATURES             Location/Qualifiers
     source          1..7663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     78..97
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    118..155
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      182..191
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             191..256
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    1508..2081
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1531..2083
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1675..1692)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1902..1921
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    2533..2757
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3221..3252
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      3279..3288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3285..4937
                     /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    5268..5389
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5305..5324)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5359..5378
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5414..5430)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5414..5430)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5427..5449)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5438..5454
                     /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(5462..5468,5469..5486,5487..5492))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5507..5528
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5645..5662)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5816..6404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5896..5915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6575..7390)
                     /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(6575..7390)
                     /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     7298..7317
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(7391..7495)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7563..7581
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7619..7641)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
       61 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcaca agcttccggc
      121 ttgtcgacga cggcggtctc cgtcgtcagg atcatccggg gtagtaataa gggcgaccgc
      181 cgccaccatg gactacaaag accatgacgg tgattataaa gatcatgaca tcgattacaa
      241 ggatgacgat gataaaggat ccggccagcc tggcaatggc agcgcctttc tgctggcccc
      301 taatggctct cacgcccccg atcacgacgt gacccaggaa cgggatgaag tgtgggtcgt
      361 gggcatgggc atcgtgatga gcctgatcgt gctggccatc gtgttcggca acgtgctcgt
      421 gatcaccgct atcgccaagt tcgagcggct gcagaccgtg accaactact tcatcacctc
      481 cctggcctgc gccgacctcg tgatgggact ggctgtggtg ccttttggag ccgcccacat
      541 cctgatgaag atgtggacct tcggcaactt ttggtgcgag ttctggacca gcatcgacgt
      601 gctgtgcgtg accgcctcca tcgagacact gtgtgtgatc gccgtggaca gatacttcgc
      661 catcaccagc cccttcaagt accagagcct gctgaccaag aacaaggcca gagtgatcat
      721 tctgatggtg tggatcgtgt ccggcctgac cagcttcctg cccatccaga tgcactggta
      781 cagagccacc caccaggaag ccatcaactg ctacgccaac gaaacctgct gcgacttctt
      841 cacaaaccag gcctacgcca ttgcctccag catcgtgtcc ttctacgtgc ccctcgtgat
      901 tatggtgttc gtgtacagcc gggtgttcca ggaagctaag cggcagctgc agaagatcga
      961 caagagcgag ggccggttcc acgtgcagaa cctgagccag gtggaacagg acggcagaac
     1021 aggccacggc ctgcggagaa gcagcaagtt ctgcctgaaa gagcacaagg ccctgaaaac
     1081 cctgggcatc atcatgggca ccttcaccct gtgctggctg cccttcttca tcgtgaacat
     1141 cgtgcacgtg atccaggaca acctgatccg gaaagaggtg tacatcctgc tgaactggat
     1201 cggctacgtg aacagcggct tcaaccccct gatctactgc agaagccccg acttccggat
     1261 cgccttccag gaactgctgt gcctgagaag aagcagcctg aaggcctacg gcaacggcta
     1321 cagcagcaac ggcaataccg gcgagcagag cggctaccat gtggaacagg aaaaagaaaa
     1381 caagctgctg tgcgaggacc tgcccggcac cgaggatttt gtgggccacc agggaaccgt
     1441 gcccagcgac aacatcgaca gccagggcag aaactgcagc accaacgaca gcctgctgtg
     1501 agctagcccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag
     1561 gccggtgtgc gtttgtctat atgtgatttt ccaccatatt gccgtctttt ggcaatgtga
     1621 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg
     1681 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt
     1741 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca
     1801 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc
     1861 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat
     1921 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc
     1981 ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga
     2041 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatgaaaac cttcaacatc
     2101 tctcagcagg atctggagct ggtggaggtc gccactgaga agatcaccat gctctatgag
     2161 gacaacaagc accatgtcgg ggcggccatc aggaccaaga ctggggagat catctctgct
     2221 gtccacattg aagcctacat tggcagggtc actgtctgtg ctgaagccat tgccattggg
     2281 tctgctgtga gcaacgggca gaaggacttt gacaccattg tggctgtcag gcacccctac
     2341 tctgatgagg tggacagatc catcagggtg gtcagcccct gtggcatgtg tagagagctg
     2401 atctctgact atgctcctga ctgctttgtg ctcattgaga tgaatggcaa gctggtcaaa
     2461 accaccattg aggaactcat ccccctcaag tacaccagga actaatctcg ttcgaaaatc
     2521 atcgcggccc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2581 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2641 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2701 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggcac
     2761 ttttatgagg gacagccccc cccccaaagc ccccagggat gtaattacgt ccctcccccg
     2821 ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc
     2881 ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc
     2941 ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc tggggggata cggggaaaag
     3001 gacgcgtgca ccagacagtg acgtcagctg ccagatccca tggccgtcat actgtgacgt
     3061 ctttcagaca gcaccagaca gtgacgtcag ctgccagatc ccatggccgt catactgtga
     3121 cgtctttcag acaccccatt gacgtcaatg ggagaacggt acctgagctc gctagcctcg
     3181 aggatatcaa gatctggcct cggcggccaa gcttagacac tagagggtat ataatggaag
     3241 ctcgacttcc agcttggcaa tccggtactg ttggtaaagc caccatggaa gatgccaaaa
     3301 acattaagaa gggcccagcg ccattctacc cactcgaaga cgggaccgcc ggcgagcagc
     3361 tgcacaaagc catgaagcgc tacgccctgg tgcccggcac catcgccttt accgacgcac
     3421 atatcgaggt ggacattacc tacgccgagt acttcgagat gagcgttcgg ctggcagaag
     3481 ctatgaagcg ctatgggctg aatacaaacc atcggatcgt ggtgtgcagc gagaatagct
     3541 tgcagttctt catgcccgtg ttgggtgccc tgttcatcgg tgtggctgtg gccccagcta
     3601 acgacatcta caacgagcgc gagctgctga acagcatggg catcagccag cccaccgtcg
     3661 tattcgtgag caagaaaggg ctgcaaaaga tcctcaacgt gcaaaagaag ctaccgatca
     3721 tacaaaagat catcatcatg gatagcaaga ccgactacca gggcttccaa agcatgtaca
     3781 ccttcgtgac ttcccatttg ccacccggct tcaacgagta cgacttcgtg cccgagagct
     3841 tcgaccggga caaaaccatc gccctgatca tgaacagtag tggcagtacc ggattgccca
     3901 agggcgtagc cctaccgcac cgcaccgctt gtgtccgatt cagtcatgcc cgcgacccca
     3961 tcttcggcaa ccagatcatc cccgacaccg ctatcctcag cgtggtgcca tttcaccacg
     4021 gcttcggcat gttcaccacg ctgggctact tgatctgcgg ctttcgggtc gtgctcatgt
     4081 accgcttcga ggaggagcta ttcttgcgca gcttgcaaga ctataagatt caatctgccc
     4141 tgctggtgcc cacactattt agcttcttcg ctaagagcac tctcatcgac aagtacgacc
     4201 taagcaactt gcacgagatc gccagcggcg gggcgccgct cagcaaggag gtaggtgagg
     4261 ccgtggccaa acgcttccac ctaccaggca tccgccaggg ctacggcctg acagaaacaa
     4321 ccagcgccat tctgatcacc cccgaagggg acgacaagcc tggcgcagta ggcaaggtgg
     4381 tgcccttctt cgaggctaag gtggtggact tggacaccgg caagacactg ggtgtgaacc
     4441 agcgcggcga gctgtgcgtc cgtggcccca tgatcatgag cggctacgtt aacaaccccg
     4501 aggctacaaa cgctctcatc gacaaggacg gctggctgca cagcggcgac atcgcctact
     4561 gggacgagga cgagcacttc ttcatcgtgg accggctgaa gagcctgatc aaatacaagg
     4621 gctaccaggt agccccagcc gaactggaga gcatcctgct gcaacacccc aacatcttcg
     4681 acgccggggt cgccggcctg cccgacgacg atgccggcga gctgcccgcc gcagtcgtcg
     4741 tgctggaaca cggtaaaacc atgaccgaga aggagatcgt ggactatgtg gccagccagg
     4801 ttacaaccgc caagaagctg cgcggtggtg ttgtgttcgt ggacgaggtg cctaaaggac
     4861 tgaccggcaa gttggacgcc cgcaagatcc gcgagattct cattaaggcc aagaagggcg
     4921 gcaagatcgc cgtgtaataa taccggtcac cctccgtacc aacgacgcat ctgattaaag
     4981 ggggtacctt atgttcacaa ctggcgtgaa tgtaactgta ttcagcgatg acccacacag
     5041 gcatagagtg tctgctatta ataactatgc tcaaaaattg tgtaccttta gctttttaat
     5101 ttgtaaaggg gttaataagg aatatttgat gtatagtgcc ttgactagaa tctagataac
     5161 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     5221 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     5281 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5341 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag gcgcgccgtt
     5401 aacgatatcc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5461 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5521 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5581 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5641 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5701 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5761 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5821 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5881 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5941 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6001 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6061 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6121 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6181 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6241 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6301 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6361 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6421 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6481 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6541 tctaaagtat atatgagtaa acttggtctg acagttagaa aaactcatcg agcatcaaat
     6601 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     6661 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     6721 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     6781 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     6841 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     6901 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     6961 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     7021 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     7081 tcccagggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     7141 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     7201 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     7261 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     7321 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     7381 tatggctcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7441 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7501 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     7561 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     7621 tctgacacat gcagctcccg gagacggtca cagcttgtct gta
//
