LOCUS       sequence-255134-        7663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes a donor vector for recombination with the Bxb1 recombinase 
            with variant G102H 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_G102H_ADRB2_LucReporter
FEATURES             Location/Qualifiers
     source          1..7663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     63..82
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    103..140
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      167..176
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             176..241
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    1493..2066
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1516..2068
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1660..1677)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1887..1906
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    2518..2742
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3206..3237
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      3264..3273
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3270..4922
                     /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    5253..5374
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5290..5309)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5344..5363
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5399..5415)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5399..5415)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5412..5434)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5423..5439
                     /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(5447..5453,5454..5471,5472..5477))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5492..5513
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5630..5647)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5801..6389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5881..5900)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6560..7375)
                     /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(6560..7375)
                     /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     7283..7302
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(7376..7480)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7548..7566
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7604..7626)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
       61 tgcggcatca gagcagattg tactgagagt gcacaagctt ccggcttgtc gacgacggcg
      121 gtctccgtcg tcaggatcat ccggggtagt aataagggcg accgccgcca ccatggacta
      181 caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgataa
      241 aggatccggc cagcctggca atggcagcgc ctttctgctg gcccctaatg gctctcacgc
      301 ccccgatcac gacgtgaccc aggaacggga tgaagtgtgg gtcgtgggca tgggcatcgt
      361 gatgagcctg atcgtgctgg ccatcgtgtt cggcaacgtg ctcgtgatca ccgctatcgc
      421 caagttcgag cggctgcaga ccgtgaccaa ctacttcatc acctccctgg cctgcgccga
      481 cctcgtgatg ggactggctg tggtgccttt tggagccgcc cacatcctga tgaagatgtg
      541 gaccttccac aacttttggt gcgagttctg gaccagcatc gacgtgctgt gcgtgaccgc
      601 ctccatcgag acactgtgtg tgatcgccgt ggacagatac ttcgccatca ccagcccctt
      661 caagtaccag agcctgctga ccaagaacaa ggccagagtg atcattctga tggtgtggat
      721 cgtgtccggc ctgaccagct tcctgcccat ccagatgcac tggtacagag ccacccacca
      781 ggaagccatc aactgctacg ccaacgaaac ctgctgcgac ttcttcacaa accaggccta
      841 cgccattgcc tccagcatcg tgtccttcta cgtgcccctc gtgattatgg tgttcgtgta
      901 cagccgggtg ttccaggaag ctaagcggca gctgcagaag atcgacaaga gcgagggccg
      961 gttccacgtg cagaacctga gccaggtgga acaggacggc agaacaggcc acggcctgcg
     1021 gagaagcagc aagttctgcc tgaaagagca caaggccctg aaaaccctgg gcatcatcat
     1081 gggcaccttc accctgtgct ggctgccctt cttcatcgtg aacatcgtgc acgtgatcca
     1141 ggacaacctg atccggaaag aggtgtacat cctgctgaac tggatcggct acgtgaacag
     1201 cggcttcaac cccctgatct actgcagaag ccccgacttc cggatcgcct tccaggaact
     1261 gctgtgcctg agaagaagca gcctgaaggc ctacggcaac ggctacagca gcaacggcaa
     1321 taccggcgag cagagcggct accatgtgga acaggaaaaa gaaaacaagc tgctgtgcga
     1381 ggacctgccc ggcaccgagg attttgtggg ccaccaggga accgtgccca gcgacaacat
     1441 cgacagccag ggcagaaact gcagcaccaa cgacagcctg ctgtgagcta gccccctctc
     1501 cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     1561 tctatatgtg attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     1621 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     1681 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
     1741 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
     1801 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
     1861 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
     1921 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
     1981 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
     2041 ttttcctttg aaaaacacga tgataatatg aaaaccttca acatctctca gcaggatctg
     2101 gagctggtgg aggtcgccac tgagaagatc accatgctct atgaggacaa caagcaccat
     2161 gtcggggcgg ccatcaggac caagactggg gagatcatct ctgctgtcca cattgaagcc
     2221 tacattggca gggtcactgt ctgtgctgaa gccattgcca ttgggtctgc tgtgagcaac
     2281 gggcagaagg actttgacac cattgtggct gtcaggcacc cctactctga tgaggtggac
     2341 agatccatca gggtggtcag cccctgtggc atgtgtagag agctgatctc tgactatgct
     2401 cctgactgct ttgtgctcat tgagatgaat ggcaagctgg tcaaaaccac cattgaggaa
     2461 ctcatccccc tcaagtacac caggaactaa tctcgttcga aaatcatcgc ggcccgactg
     2521 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2581 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2641 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2701 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcactttta tgagggacag
     2761 cccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc
     2821 gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt
     2881 gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc
     2941 gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaggacgc gtgcaccaga
     3001 cagtgacgtc agctgccaga tcccatggcc gtcatactgt gacgtctttc agacagcacc
     3061 agacagtgac gtcagctgcc agatcccatg gccgtcatac tgtgacgtct ttcagacacc
     3121 ccattgacgt caatgggaga acggtacctg agctcgctag cctcgaggat atcaagatct
     3181 ggcctcggcg gccaagctta gacactagag ggtatataat ggaagctcga cttccagctt
     3241 ggcaatccgg tactgttggt aaagccacca tggaagatgc caaaaacatt aagaagggcc
     3301 cagcgccatt ctacccactc gaagacggga ccgccggcga gcagctgcac aaagccatga
     3361 agcgctacgc cctggtgccc ggcaccatcg cctttaccga cgcacatatc gaggtggaca
     3421 ttacctacgc cgagtacttc gagatgagcg ttcggctggc agaagctatg aagcgctatg
     3481 ggctgaatac aaaccatcgg atcgtggtgt gcagcgagaa tagcttgcag ttcttcatgc
     3541 ccgtgttggg tgccctgttc atcggtgtgg ctgtggcccc agctaacgac atctacaacg
     3601 agcgcgagct gctgaacagc atgggcatca gccagcccac cgtcgtattc gtgagcaaga
     3661 aagggctgca aaagatcctc aacgtgcaaa agaagctacc gatcatacaa aagatcatca
     3721 tcatggatag caagaccgac taccagggct tccaaagcat gtacaccttc gtgacttccc
     3781 atttgccacc cggcttcaac gagtacgact tcgtgcccga gagcttcgac cgggacaaaa
     3841 ccatcgccct gatcatgaac agtagtggca gtaccggatt gcccaagggc gtagccctac
     3901 cgcaccgcac cgcttgtgtc cgattcagtc atgcccgcga ccccatcttc ggcaaccaga
     3961 tcatccccga caccgctatc ctcagcgtgg tgccatttca ccacggcttc ggcatgttca
     4021 ccacgctggg ctacttgatc tgcggctttc gggtcgtgct catgtaccgc ttcgaggagg
     4081 agctattctt gcgcagcttg caagactata agattcaatc tgccctgctg gtgcccacac
     4141 tatttagctt cttcgctaag agcactctca tcgacaagta cgacctaagc aacttgcacg
     4201 agatcgccag cggcggggcg ccgctcagca aggaggtagg tgaggccgtg gccaaacgct
     4261 tccacctacc aggcatccgc cagggctacg gcctgacaga aacaaccagc gccattctga
     4321 tcacccccga aggggacgac aagcctggcg cagtaggcaa ggtggtgccc ttcttcgagg
     4381 ctaaggtggt ggacttggac accggcaaga cactgggtgt gaaccagcgc ggcgagctgt
     4441 gcgtccgtgg ccccatgatc atgagcggct acgttaacaa ccccgaggct acaaacgctc
     4501 tcatcgacaa ggacggctgg ctgcacagcg gcgacatcgc ctactgggac gaggacgagc
     4561 acttcttcat cgtggaccgg ctgaagagcc tgatcaaata caagggctac caggtagccc
     4621 cagccgaact ggagagcatc ctgctgcaac accccaacat cttcgacgcc ggggtcgccg
     4681 gcctgcccga cgacgatgcc ggcgagctgc ccgccgcagt cgtcgtgctg gaacacggta
     4741 aaaccatgac cgagaaggag atcgtggact atgtggccag ccaggttaca accgccaaga
     4801 agctgcgcgg tggtgttgtg ttcgtggacg aggtgcctaa aggactgacc ggcaagttgg
     4861 acgcccgcaa gatccgcgag attctcatta aggccaagaa gggcggcaag atcgccgtgt
     4921 aataataccg gtcactccgc atcgacccga cgcatctgat taaagggggt accttatgtt
     4981 cacaactggc gtgaatgtaa ctgtattcag cgatgaccca cacaggcata gagtgtctgc
     5041 tattaataac tatgctcaaa aattgtgtac ctttagcttt ttaatttgta aaggggttaa
     5101 taaggaatat ttgatgtata gtgccttgac tagaatctag ataactgatc ataatcagcc
     5161 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     5221 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     5281 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5341 gttgtggttt gtccaaactc atcaatgtat cttaggcgcg ccgttaacga tatccatggt
     5401 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5461 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5521 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5581 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5641 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5701 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5761 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5821 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5881 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5941 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6001 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6061 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6121 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6181 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6241 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6301 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6361 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6421 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6481 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6541 agtaaacttg gtctgacagt tagaaaaact catcgagcat caaatgaaac tgcaatttat
     6601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     6661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     6721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     6781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     6841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     6901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     6961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     7021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttccca gggatcgcag
     7081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     7141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     7201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     7261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     7321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcatactct
     7381 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7441 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7501 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     7561 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     7621 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gag
//
