LOCUS       sequence-142448-        8243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a polyspecific aminoacyl-tRNA synthetase and four copies of 
            amber suppression tRNAs for unnatural amino acid incorporation in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8243)
  AUTHORS   Chen ZJ, Ren W, Wright QE, Ai HW
  TITLE     Genetically encoded fluorescent probe for the selective detection of
            peroxynitrite.
  JOURNAL   J Am Chem Soc. 2013 Oct 9;135(40):14940-3. doi: 10.1021/ja408011q. 
            Epub 2013 Sep 26.
  PUBMED    24059533
REFERENCE   2  (bases 1 to 8243)
  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"; doi: "10.1021/ja408011q";
            journalName: "J Am Chem Soc"; date: "2013-10-9- 9"; volume: "135"; 
            issue: "40"; pages: "14940-3"
COMMENT     Sequence Label: pMAH-POLY
FEATURES             Location/Qualifiers
     source          1..8243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     505..529
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        739..757
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     739..756
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     770..789
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      822..831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2125..2142)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2131..2355
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2401..2829
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2488..2507)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2698..2719
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2838..2858)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2843..3172
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3023..3158
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3085..3104
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3221..4246
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3221..4246
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4376..4497
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4413..4432)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4467..4486
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4546..4562)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4546..4562)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4559..4581)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4570..4586
                     /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(4594..4600,4601..4618,4619..4624))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4639..4660
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4777..4794)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4948..5536)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5028..5047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5707..6498,6499..6567))
                     /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     6330..6349
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6568..6672)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(6796..6886)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     promoter        complement(6998..7246)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(7535..7625)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     promoter        complement(7737..7985)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg
      541 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg
      601 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag
      661 agtctatagg cccaccccct tggcttcgtt agaacgcggc tacaattaat acataacctt
      721 atgtatcata cacatacgat ttaggtgaca ctatagaata acatccactt tgcctttctc
      781 tccacaggtg tccactccca ggtccaactg cacggaagct tgccaccatg gcaagcagta
      841 acttgattaa acaattgcaa gagcgggggc tggtagccca ggtgacggac gaggaagcgc
      901 tagcagagcg actggcgcaa ggcccgatcg cactcatttg tggcttcgat cctaccgctg
      961 acagcttgca tttggggcat cttgttccat tgttatgcct gaaacgcttc cagcaggcgg
     1021 gccacaagcc ggttgcgctg gtaggcggcg cgacgggtct gattggcgac ccgagcttca
     1081 aagctgccga gcgtaagctg aacaccgaag aaactgttca ggagtgggtg gacaaaatcc
     1141 gtaagcaggt tgccccgttc ctcgatttcg actgtggaga aaactctgct atcgcggcca
     1201 ataattatga ctggttcggc aatatgaatg tgctgacctt cctgcgcgat attggcaaac
     1261 acttctccgt taaccagatg atcaacaaag aagcggttaa gcagcgtctc aaccgtgaag
     1321 atcaggggat ttcgttcact gagttttcct acaacctgct gcagggttat tcgatggcct
     1381 gtgctaacaa acagtacggt gtggtgctgc aaattggtgg ttctgaccag tggggtaaca
     1441 tcacttctgg tatcgacctg acccgtcgtc tgcatcagaa tcaggtgttt ggcctgaccg
     1501 ttccgctgat cactaaagca gatggcacca aatttggtaa aactgaaggc ggcgcagtct
     1561 ggttggatcc gaagaaaacc agcccgtaca aattctacca gttctggatc aacactgcgc
     1621 gtgccgacgt ttaccgcttc ctgaagttct tcacctttat gagcattgaa gagatcaacg
     1681 ccctggaaga agaagataaa aacagcggta aagcaccgcg cgcccagtat gtactggcgg
     1741 agcaggtgac tcgtctggtt cacggtgaag aaggtttaca ggcggcaaaa cgtattaccg
     1801 aatgcctgtt cagcggttct ttgagtgcgc tgagtgaagc ggacttcgaa cagctggcgc
     1861 aggacggcgt accgatggtt gagatggaaa agggcgcaga cctgatgcag gcactggtcg
     1921 attctgaact gcaaccttcc cgtggtcagg cacgtaaaac tatcgcctcc aatgccatca
     1981 ccattaacgg tgaaaaacag tccgatcctg aatacttctt taaagaagaa gatcgtctgt
     2041 ttggtcgttt taccttactg cgtcgcggta aaaagaatta ctgtctgatt tgctggaaag
     2101 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2161 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2221 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2281 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     2341 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     2401 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2461 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2521 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2581 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2641 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2701 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2761 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2821 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2881 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2941 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3001 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3061 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     3121 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     3181 gggagcttgt atatccattt tcggatctga tcagcacgtg atgaaaaagc ctgaactcac
     3241 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     3301 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     3361 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     3421 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct
     3481 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     3541 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     3601 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     3661 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     3721 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     3781 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     3841 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     3901 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     3961 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     4021 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     4081 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     4141 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     4201 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tgctacgaga
     4261 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4321 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4381 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4441 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4501 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4561 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4621 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4681 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4741 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4801 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4861 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4921 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4981 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5041 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5101 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5161 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5221 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5281 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5341 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5401 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5461 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5521 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5581 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5641 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5701 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5761 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5821 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5881 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5941 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6001 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6061 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6121 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6181 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6241 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6301 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6361 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6421 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6481 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6541 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6601 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6661 aggggttccg cgcacatttc cccgactcga ctcgctagtg ttccaaaaaa tggtggggga
     6721 cggattcgaa ccgccgaacc caaagggagc ggatttagag tccgcggcgt ttggccactt
     6781 cgctacccca ccagagtggt ctcatacaga acttataaga ttcccaaatc caaagacatt
     6841 tcacgtttat ggtgatttcc cagaacacat agcgacatgc aaatatgaat tgatgactgc
     6901 ctgatggaga aaaaatggtg ggggacggat tcgaaccgcc gaacccaaag ggagcggatt
     6961 tagagtccgc ggcgtttggc cacttcgcta ccccaccggt gtttcgtcct ttccacaaga
     7021 tatataaagc caagaaatcg aaatactttc aagttacggt aagcatatga tagtccattt
     7081 taaaacataa ttttaaaact gcaaactacc caagaaatta ttactttcta cgtcacgtat
     7141 tttgtactaa tatctttgtg tttacagtca aattaattct aattatctct ctaacagcct
     7201 tgtatcgtat atgcaaatat gaaggaatca tgggaaatag gccctcttcc tgcccgacct
     7261 tcgcgatgta cgggccagat atacgccaat attggccatt agccatatta ttcattggtt
     7321 atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata
     7381 tgtacattta tattggctca tgtccaatat gaccgccatg ttggctcgac tcgctagtgt
     7441 tccaaaaaat ggtgggggac ggattcgaac cgccgaaccc aaagggagcg gatttagagt
     7501 ccgcggcgtt tggccacttc gctaccccac cagagtggtc tcatacagaa cttataagat
     7561 tcccaaatcc aaagacattt cacgtttatg gtgatttccc agaacacata gcgacatgca
     7621 aatatgaatt gatgactgcc tgatggagaa aaaatggtgg gggacggatt cgaaccgccg
     7681 aacccaaagg gagcggattt agagtccgcg gcgtttggcc acttcgctac cccaccggtg
     7741 tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca agttacggta
     7801 agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc aagaaattat
     7861 tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa attaattcta
     7921 attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat gggaaatagg
     7981 ccctcttcct gcccgacctt cgcgatgtac gggccagata tacgccaata ttggccatta
     8041 gccatattat tcattggtta tatagcataa atcaatattg gctattggcc attgcatacg
     8101 ttgtatctat atcataatat gtacatttat attggctcat gtccaatatg accgccatgt
     8161 tggctcgagc attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     8221 catagcccat atatggagtt ccg
//
