LOCUS       sequence-388871-        7708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express azidophenylalanine-specific variant of E. coli TyrRS 
            (pAzpaRS) and its cognate amber suppressor tRNA to allow UAG codon 
            to be translated into azidophenylalanine..
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: pcpAzpaRS_v2
FEATURES             Location/Qualifiers
     source          1..7708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1861..1880)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     enhancer        1977..2356
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2357..2560
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2511..2531
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2562..2580
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2583..2601
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2611..2635
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2831..2848)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2837..3061
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3107..3535
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3194..3213)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3404..3425
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3544..3564)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3549..3878
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3729..3864
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3791..3810
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5213..5239
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5381..5502
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5418..5437)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5472..5491
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5551..5567)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5551..5567)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5564..5586)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5575..5591
                     /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(5599..5605,5606..5623,5624..5629))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5644..5665
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5782..5799)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5953..6541)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6033..6052)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6712..7503,7504..7572))
                     /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     7335..7354
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7573..7677)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcggc cctttcgtct tcaagaattc agcgagtgtt
      241 aaccctgcct agcgctccgg tttttctgtg ctgaacctca ggggacgccg acacacgtac
      301 acgtcggagg ggtagcgaag tggctaaacg cggcggactc taaatccgct ccctttgggt
      361 tcggcggttc gaatccgtcc ccctccagac aagtgcggtt tttttctcca gctcccgcca
      421 gcagactgga gcgctccggt ttttctgtgc tgaacctcag gggacgccga cacacgtaca
      481 cgtcggaggg gtagcgaagt ggctaaacgc ggcggactct aaatccgctc cctttgggtt
      541 cggcggttcg aatccgtccc cctccagaca agtgcggttt ttttctccag ctcccgccag
      601 cttcctggag tgctccggtt tttctgtgct gaacctcagg ggacgccgac acacgtacac
      661 gtcggagggg tagcgaagtg gctaaacgcg gcggactcta aatccgctcc ctttgggttc
      721 ggcggttcga atccgtcccc ctccagacaa gtgcggtttt tttctccagc tcccgcgact
      781 acgatattcg cgcagaagct tagcgagtgt taaccctgcc tagcgctccg gtttttctgt
      841 gctgaacctc aggggacgcc gacacacgta cacgtcggag gggtagcgaa gtggctaaac
      901 gcggcggact ctaaatccgc tccctttggg ttcggcggtt cgaatccgtc cccctccaga
      961 caagtgcggt ttttttctcc agctcccgcc agcagactgg agcgctccgg tttttctgtg
     1021 ctgaacctca ggggacgccg acacacgtac acgtcggagg ggtagcgaag tggctaaacg
     1081 cggcggactc taaatccgct ccctttgggt tcggcggttc gaatccgtcc ccctccagac
     1141 aagtgcggtt tttttctcca gctcccgcca gcttcctgga gcgctccggt ttttctgtgc
     1201 tgaacctcag gggacgccga cacacgtaca cgtcggaggg gtagcgaagt ggctaaacgc
     1261 ggcggactct aaatccgctc cctttgggtt cggcggttcg aatccgtccc cctccagaca
     1321 agtgcggttt ttttctccag ctcccgcgac tacgatattc gcgcaggaat tcagcgagtg
     1381 ttaaccctgc ctagcgctcc ggtttttctg tgctgaacct caggggacgc cgacacacgt
     1441 acacgtcgga ggggtagcga agtggctaaa cgcggcggac tctaaatccg ctccctttgg
     1501 gttcggcggt tcgaatccgt ccccctccag acaagtgcgg tttttttctc cagctcccgc
     1561 cagcagactg gagcgctccg gtttttctgt gctgaacctc aggggacgcc gacacacgta
     1621 cacgtcggag gggtagcgaa gtggttaaac gcggcggact ctaaatccgc tccctttggg
     1681 ttcggcggtt cgaatccgtc cccctccaga caagtgcggt ttttttctcc agctcccgcc
     1741 agcttcctgg agcgctccgg tttttctcca gctcccgcga ctacgatatt cgcgcaggga
     1801 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     1861 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     1921 cttgtttcgg cgtgggtatg gtggcaggcc cgatgtacgg gccagatata cgcgttgaca
     1981 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     2041 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     2101 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     2161 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     2221 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     2281 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     2341 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     2401 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     2461 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     2521 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga
     2581 tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg
     2641 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
     2701 gactctagcg tttaaactta agcttggtac cgagctcgga tccactagtc cagtgtggtg
     2761 gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc
     2821 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2881 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2941 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3001 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3061 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3121 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3181 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3241 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3301 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3361 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3421 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3481 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3541 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3601 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3661 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3721 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3781 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3841 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     3901 ccattttcgg atctgatcag caccacacag aattcccacc atggcaagca gtaacttgat
     3961 taaacaattg caagagcggg ggctggtagc ccaggtgacg gacgaggaag cgttagcaga
     4021 gcgactggcg caaggcccga tcgcgctctt gtgcggcttc gatcctaccg ctgacagctt
     4081 gcatttgggg catcttgttc cattgttatg cctgaaacgc ttccagcagg cgggccacaa
     4141 gccggttgcg ctggtaggcg gcgcgacggg tctgattggc gacccgagct tcaaagctgc
     4201 cgagcgtaag ctgaacaccg aagaaactgt tcaggagtgg gtggacaaaa tccgtaagca
     4261 ggttgccccg ttcctcgatt tcgactgtgg agaaaactct gctatcgcgg cgaacaacta
     4321 tgactggttc ggcaatatga atgtgctgac cttcctgcgc gatattggca aacacttctc
     4381 cgttaaccag atgatcaaca aagaagcggt taagcagcgt ctcaaccgtg aagatcaggg
     4441 gatttcgttc actgagtttt cctacaacct gttgcagggt tattccatgg cctgtgcgaa
     4501 caaacagtac ggtgtggtgc tgcaaattgg tggttctgac cagtggggta acatcacttc
     4561 tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg tttggcctga ccgttccgct
     4621 gatcactaaa gcagatggca ccaaatttgg taaaactgaa ggcggcgcag tctggttgga
     4681 tccgaagaaa accagcccgt acaaattcta ccagttctgg atcaacactg cggatgccga
     4741 cgtttaccgc ttcctgaagt tcttcacctt tatgagcatt gaagagatca acgccctgga
     4801 agaagaagat aaaaacagcg gtaaagcacc gcgcgcccag tatgtactgg cggagcaggt
     4861 gactcgtctg gttcacggtg aagaaggttt acaggcggca aaacgtatta ccgaatgcct
     4921 gttcagcggt tctttgagtg cgctgagtga agcggacttc gaacagctgg cgcaggacgg
     4981 cgtaccgatg gttgagatgg aaaagggcgc agacctgatg caggcactgg tcgattctga
     5041 actgcaacct tcccgtggtc aggcacgtaa aactatcgcc tccaatgcca tcaccattaa
     5101 cggtgaaaaa cagtccgatc ctgaatactt ctttaaagaa gaagatcgtc tgtttggtcg
     5161 ttttacctta ctgcgtcgcg gtaaaaagaa ttactgtctg atttgctgga aatacccata
     5221 cgatgttcca gattacgctt aactcgagtg tgtggtgcta cgagatttcg attccaccgc
     5281 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5341 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5401 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5461 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5521 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5581 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5641 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5701 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5761 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5821 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5881 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5941 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6001 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6061 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6121 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6181 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6241 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6301 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6361 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6421 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6481 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6541 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6601 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6661 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6721 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6781 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6841 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6901 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6961 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7021 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7081 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7141 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7201 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7261 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7321 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7381 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7441 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7501 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7561 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7621 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7681 atttccccga aaagtgccac ctgacgtc
//
