LOCUS       sequence-386704-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express Methanosarcina mazei PylRS(Y384F Y306A) and allow UAG 
            codon to be translated into Z-Lys or Azido-Z-Lys when used together 
            with the pU6tRNAPyl plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  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 6354)
  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: pcPylRS(Y384F Y306A)
FEATURES             Location/Qualifiers
     source          1..6354
                     /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"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2362..2379)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2368..2592
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2638..3066
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2725..2744)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2935..2956
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3075..3095)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3080..3409
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3260..3395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3322..3341
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3457..3504
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3523..3897
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    4027..4148
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4064..4083)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4118..4137
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4197..4213)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4197..4213)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4210..4232)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4221..4237
                     /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(4245..4251,4252..4269,4270..4275))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4290..4311
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4428..4445)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4599..5187)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4679..4698)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5358..6149,6150..6218))
                     /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     5981..6000
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6219..6323)
                     /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 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 accatggata aaaaaccact aaacactctg atatctgcaa ccgggctctg gatgtccagg
      961 accggaacaa ttcataaaat aaaacaccac gaagtctctc gaagcaaaat ctatattgaa
     1021 atggcatgcg gagaccacct tgttgtaaac aactccagga gcagcaggac tgcaagagcg
     1081 ctcaagcacc acaaatacag gaagacctgc aaacgctgca gggtttcgga tgaggatctc
     1141 aataagttcc tcacaaaggc aaacgaagac cagacaagcg taaaagtcaa ggtcgtttct
     1201 gcccctacca gaacgaaaaa ggcaatgcca aaatccgttg cgagagcccc gaaacctctt
     1261 gagaatacag aagcggcaca ggctcaacct tctggatcta aattttcacc tgcgataccg
     1321 gtttccaccc aagagtcagt ttctgtcccg gcatctgttt caacatcaat atcaagcatt
     1381 tctacaggag caactgcatc cgcactggta aaagggaata cgaaccccat tacatccatg
     1441 tctgcccctg ttcaggcaag tgcccccgca cttacgaaga gccagactga caggcttgaa
     1501 gtcctgttaa acccaaaaga tgagatttcc ctgaattccg gcaagccttt cagggagctt
     1561 gagtccgaat tgctctctcg cagaaaaaaa gacctgcagc agatctacgc ggaagaaagg
     1621 gagaattatc tggggaaact cgagcgtgaa attaccaggt tctttgtgga caggggtttt
     1681 ctggaaataa aatccccgat cctgatccct cttgagtata tcgaaaggat gggcattgat
     1741 aatgataccg aactttcaaa acagatcttc agggttgaca agaacttctg cctgagaccc
     1801 atgcttgctc caaaccttgc caactacctg cgcaagcttg acagggccct gcctgatcca
     1861 ataaaaattt ttgaaatagg cccatgctac agaaaagagt ccgacggcaa agaacacctc
     1921 gaagagttta ccatgctgaa cttctgccag atgggatcgg gatgcacacg ggaaaatctt
     1981 gaaagcataa ttacagactt cctgaaccac ctgggaattg atttcaagat cgtaggcgat
     2041 tcctgcatgg tctttgggga tacccttgat gtaatgcacg gagacctgga actttcctct
     2101 gcagtagtcg gacccatacc gcttgaccgg gaatggggta ttgataaacc ctggataggg
     2161 gcaggtttcg ggctcgaacg ccttctcaag gttaaacacg actttaaaaa tatcaagaga
     2221 gctgcaaggt ccgggtctta ctataacggg atttctacca acctgtaagg atccactagt
     2281 ccagtgtggt ggaattctgc agatatccag cacagtggcg gccgctcgag tctagagggc
     2341 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2401 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2461 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     2521 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     2581 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     2641 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2701 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2761 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2821 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2881 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2941 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3001 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3061 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     3121 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3181 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3241 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3301 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     3361 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     3421 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag
     3481 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt
     3541 gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg
     3601 ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg
     3661 accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg
     3721 tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc
     3781 cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc
     3841 gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac
     3901 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3961 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4021 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4081 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4141 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4201 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4261 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4321 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4381 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4441 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4501 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4561 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4621 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4681 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4741 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4801 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4861 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4921 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4981 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5041 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5101 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5161 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5221 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5281 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5341 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5401 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5461 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5521 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5581 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5641 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5701 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5761 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5821 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5881 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5941 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6001 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6061 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6121 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6181 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6241 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6301 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
