LOCUS       sequence-388866-        7708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express benzoylphenylalanine-specific variant of E. coli TyrRS 
            (pBpaRS) and its cognate amber suppressor tRNA to allow UAG codon to
            be translated into benzoylphenylalanine..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7708)
  AUTHORS   Hino N, Sakamoto K, Yokoyama S
  TITLE     Site-specific incorporation of unnatural amino acids into proteins 
            in mammalian cells.
  JOURNAL   Methods Mol Biol. 2012;794:215-28. doi: 
            10.1007/978-1-61779-331-8_13.
  PUBMED    21956565
REFERENCE   2  (bases 1 to 7708)
  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: "Methods Mol
            Biol."; date: "2012"; volume: "794:215-28. doi"; pages: " 
            10.1007/978-1-61779-331-8_13"
COMMENT     Sequence Label: pcpBpaRS_v2
FEATURES             Location/Qualifiers
     source          1..7708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..59)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1857..1876)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     enhancer        1973..2352
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2353..2556
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2507..2527
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2558..2576
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2579..2597
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2607..2631
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2827..2844)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2833..3057
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3103..3531
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3190..3209)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3400..3421
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3540..3560)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3545..3874
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3725..3860
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3787..3806
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5209..5235
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5377..5498
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5414..5433)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5468..5487
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5547..5563)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5547..5563)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5560..5582)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5571..5587
                     /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(5595..5601,5602..5619,5620..5625))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5640..5661
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5778..5795)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5949..6537)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6029..6048)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6708..7499,7500..7568))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7331..7350
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7569..7673)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
       61 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
      121 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
      181 ggttaggcgt tttgcgctgc ttcggccctt tcgtcttcaa gaattcagcg agtgttaacc
      241 ctgcctagcg ctccggtttt tctgtgctga acctcagggg acgccgacac acgtacacgt
      301 cggaggggta gcgaagtggc taaacgcggc ggactctaaa tccgctccct ttgggttcgg
      361 cggttcgaat ccgtccccct ccagacaagt gcggtttttt tctccagctc ccgccagcag
      421 actggagcgc tccggttttt ctgtgctgaa cctcagggga cgccgacaca cgtacacgtc
      481 ggaggggtag cgaagtggct aaacgcggcg gactctaaat ccgctccctt tgggttcggc
      541 ggttcgaatc cgtccccctc cagacaagtg cggttttttt ctccagctcc cgccagcttc
      601 ctggagtgct ccggtttttc tgtgctgaac ctcaggggac gccgacacac gtacacgtcg
      661 gaggggtagc gaagtggcta aacgcggcgg actctaaatc cgctcccttt gggttcggcg
      721 gttcgaatcc gtccccctcc agacaagtgc ggtttttttc tccagctccc gcgactacga
      781 tattcgcgca gaagcttagc gagtgttaac cctgcctagc gctccggttt ttctgtgctg
      841 aacctcaggg gacgccgaca cacgtacacg tcggaggggt agcgaagtgg ctaaacgcgg
      901 cggactctaa atccgctccc tttgggttcg gcggttcgaa tccgtccccc tccagacaag
      961 tgcggttttt ttctccagct cccgccagca gactggagcg ctccggtttt tctgtgctga
     1021 acctcagggg acgccgacac acgtacacgt cggaggggta gcgaagtggc taaacgcggc
     1081 ggactctaaa tccgctccct ttgggttcgg cggttcgaat ccgtccccct ccagacaagt
     1141 gcggtttttt tctccagctc ccgccagctt cctggagcgc tccggttttt ctgtgctgaa
     1201 cctcagggga cgccgacaca cgtacacgtc ggaggggtag cgaagtggct aaacgcggcg
     1261 gactctaaat ccgctccctt tgggttcggc ggttcgaatc cgtccccctc cagacaagtg
     1321 cggttttttt ctccagctcc cgcgactacg atattcgcgc aggaattcag cgagtgttaa
     1381 ccctgcctag cgctccggtt tttctgtgct gaacctcagg ggacgccgac acacgtacac
     1441 gtcggagggg tagcgaagtg gctaaacgcg gcggactcta aatccgctcc ctttgggttc
     1501 ggcggttcga atccgtcccc ctccagacaa gtgcggtttt tttctccagc tcccgccagc
     1561 agactggagc gctccggttt ttctgtgctg aacctcaggg gacgccgaca cacgtacacg
     1621 tcggaggggt agcgaagtgg ttaaacgcgg cggactctaa atccgctccc tttgggttcg
     1681 gcggttcgaa tccgtccccc tccagacaag tgcggttttt ttctccagct cccgccagct
     1741 tcctggagcg ctccggtttt tctccagctc ccgcgactac gatattcgcg cagggatcct
     1801 ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta
     1861 tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg
     1921 tttcggcgtg ggtatggtgg caggcccgat gtacgggcca gatatacgcg ttgacattga
     1981 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     2041 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     2101 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     2161 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2221 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     2281 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2341 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     2401 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     2461 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     2521 aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga tagagatctc
     2581 cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat cgcctggaga
     2641 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccggact
     2701 ctagcgttta aacttaagct tggtaccgag ctcggatcca ctagtccagt gtggtggaat
     2761 tctgcagata tccagcacag tggcggccgc tcgagtctag agggcccgtt taaacccgct
     2821 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2881 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2941 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3001 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     3061 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     3121 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3181 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3241 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3301 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3361 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3421 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3481 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     3541 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3601 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3661 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3721 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3781 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     3841 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     3901 tttcggatct gatcagcacc acacagaatt cccaccatgg caagcagtaa cttgattaaa
     3961 caattgcaag agcgggggct ggtagcccag gtgacggacg aggaagcgtt agcagagcga
     4021 ctggcgcaag gcccgatcgc gctcggttgc ggcttcgatc ctaccgctga cagcttgcat
     4081 ttggggcatc ttgttccatt gttatgcctg aaacgcttcc agcaggcggg ccacaagccg
     4141 gttgcgctgg taggcggcgc gacgggtctg attggcgacc cgagcttcaa agctgccgag
     4201 cgtaagctga acaccgaaga aactgttcag gagtgggtgg acaaaatccg taagcaggtt
     4261 gccccgttcc tcgatttcga ctgtggagaa aactctgcta tcgcggcgaa caactatgac
     4321 tggttcggca atatgaatgt gctgaccttc ctgcgcgata ttggcaaaca cttctccgtt
     4381 aaccagatga tcaacaaaga agcggttaag cagcgtctca accgtgaaga tcaggggatt
     4441 tcgttcactg agttttccta caacctgttg cagggttatg gcttcgcctg tgcgaacaaa
     4501 cagtacggtg tggtgctgca aattggtggt tctgaccagt ggggtaacat cacttctggt
     4561 atcgacctga cccgtcgtct gcatcagaat caggtgtttg gcctgaccgt tccgctgatc
     4621 actaaagcag atggcaccaa atttggtaaa actgaaggcg gcgcagtctg gttggatccg
     4681 aagaaaacca gcccgtacaa attctaccag ttctggatca acactgcgga tgccgacgtt
     4741 taccgcttcc tgaagttctt cacctttatg agcattgaag agatcaacgc cctggaagaa
     4801 gaagataaaa acagcggtaa agcaccgcgc gcccagtatg tactggcgga gcaggtgact
     4861 cgtctggttc acggtgaaga aggtttacag gcggcaaaac gtattaccga atgcctgttc
     4921 agcggttctt tgagtgcgct gagtgaagcg gacttcgaac agctggcgca ggacggcgta
     4981 ccgatggttg agatggaaaa gggcgcagac ctgatgcagg cactggtcga ttctgaactg
     5041 caaccttccc gtggtcaggc acgtaaaact atcgcctcca atgccatcac cattaacggt
     5101 gaaaaacagt ccgatcctga atacttcttt aaagaagaag atcgtctgtt tggtcgtttt
     5161 accttactgc gtcgcggtaa aaagaattac tgtctgattt gctggaaata cccatacgat
     5221 gttccagatt acgcttaact cgagtgtgtg gtgctacgag atttcgattc caccgccgcc
     5281 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     5341 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     5401 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5461 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     5521 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5581 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5641 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5701 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5761 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5821 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5881 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5941 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     6001 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6061 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6121 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6181 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6241 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6301 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6361 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6421 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6481 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6541 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6601 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6661 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6721 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6781 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6841 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6901 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6961 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     7021 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7081 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7141 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7201 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7261 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7321 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7381 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7441 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7501 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7561 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7621 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7681 ccccgaaaag tgccacctga cgtcgacg
//
