LOCUS       sequence-437735-        7461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PylRS (AF)/tRNA Pyl orthogonal pair for genetic code expansion that 
            allows site-specific incorporation of noncanonical amino-acids into 
            a POI using the Amber stop codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7461)
  AUTHORS   Nikic I, Estrada Girona G, Kang JH, Paci G, Mikhaleva S, Koehler C, 
            Shymanska NV, Ventura Santos C, Spitz D, Lemke EA
  TITLE     Debugging Eukaryotic Genetic Code Expansion for Site-Specific 
            Click-PAINT Super-Resolution Microscopy.
  JOURNAL   Angew Chem Int Ed Engl. 2016 Dec 23;55(52):16172-16176. doi: 
            10.1002/anie.201608284. Epub 2016 Nov 2.
  PUBMED    27804198
REFERENCE   2  (bases 1 to 7461)
  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: 
            "10.1002/anie.201608284"; journalName: "Angew Chem Int Ed Engl"; 
            date: "2016-12-23- 23"; volume: "55"; issue: "52"; pages: 
            "16172-16176"
COMMENT     Sequence Label: pCMV_NESPylRS(AF)_hU6tRNAPyl
FEATURES             Location/Qualifiers
     source          1..7461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        150..529
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        530..733
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     684..704
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     730..754
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        964..982
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     964..981
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     995..1014
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             1075..1101
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(2548..2565)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2554..2778
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2824..3252
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2911..2930)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3121..3142
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3261..3281)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3266..3596
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3447..3582
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3509..3528
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3645..4670
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3645..4670
                     /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    4800..4921
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4837..4856)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4891..4910
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4970..4986)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4970..4986)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4983..5005)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4994..5010
                     /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(5018..5024,5025..5042,5043..5048))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5063..5084
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5201..5218)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5372..5960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5452..5471)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6131..6922,6923..6991))
                     /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     6754..6773
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6992..7096)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(7186..7434)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7244..7263)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7414..7434)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gccagatata cgccaatatt ggccattagc catattattc attggttata tagcataaat
       61 caatattggc tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat
      121 tggctcatgt ccaatatgac cgccatgttg acattgatta ttgactagtt attaatagta
      181 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      241 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      301 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggactattt
      361 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      421 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      481 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      541 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      601 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      661 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      721 tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
      781 tgacctccat agaagacacc gggaccgatc cagcctccgc ggccgggaac ggtgcattgg
      841 aacgcggatt ccccgtgcca agagtgacgt aagtaccgcc tatagagtct ataggcccac
      901 ccccttggct tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
      961 acgatttagg tgacactata gaataacatc cactttgcct ttctctccac aggtgtccac
     1021 tcccaggtcc aactgcacgg aagcttcacc atggcgtgcc cggtgccgct gcagctgccg
     1081 ccgctggaac gcctgaccct ggatgataaa aaaccgctga ataccctgat ctctgctact
     1141 ggtctgtgga tgagtcgtac cggaaccatt cataaaatca aacaccacga ggttagccgt
     1201 tcgaaaatct atattgagat ggcgtgtggc gatcatctgg ttgtgaacaa tagccgctct
     1261 tctcgtacag cacgtgcact gcgtcaccac aaatatcgta aaacctgtaa acgttgccgt
     1321 gtgtccgatg aggatctgaa caaattcctg acaaaagcca atgaggacca aacaagcgtg
     1381 aaagtgaaag tcgttagcgc tcctacccgt actaaaaaag caatgccgaa atccgttgct
     1441 cgtgccccta aaccactgga aaacactgaa gcagcacagg cacagccgtc tggaagcaaa
     1501 ttctctccgg ccattcctgt ttctacccag gagtccgttt ctgttccagc aagtgtgagc
     1561 accagcatta gcagtattag caccggtgcc accgctagcg ccctggttaa aggcaatacc
     1621 aatccgatta caagcatgtc tgccccggtt caagcatcag ctccagcact gacaaaatcc
     1681 caaaccgatc gtctggaggt tctgctgaat ccgaaagacg aaatcagcct gaattccggc
     1741 aaaccgtttc gtgaactgga gagcgaactg ctgtcacgtc gtaaaaaaga cctgcaacaa
     1801 atctatgccg aagaacgtga gaactatctg gggaaactgg aacgtgaaat cacccgcttt
     1861 ttcgtggatc gtggctttct ggagatcaaa tccccgattc tgattcctct ggagtatatc
     1921 gagcgtatgg gcatcgacaa tgataccgaa ctgagcaaac aaattttccg tgtggataaa
     1981 aacttctgtc tgcgccctat gctagcacca aatctggcta actatctgcg caaactggac
     2041 cgtgccctgc ctgatcctat caaaatcttc gagatcggcc cgtgttatcg taaagagtcc
     2101 gacggtaaag aacatctgga ggagtttacc atgctgaact tttgccaaat gggttcaggt
     2161 tgtactcgtg agaacctgga aagcatcatc accgattttc tgaaccacct gggcattgac
     2221 ttcaaaattg tgggcgacag ctgtatggtg tttggcgaca ccctggatgt catgcacggc
     2281 gacctggaac tgtctagtgc cgttgtgggc ccaatcccgc tggatcgtga gtggggtatc
     2341 gacaaacctt ggatcggtgc gggttttggt ctggagcgtc tgctgaaagt aaaacacgac
     2401 ttcaagaaca tcaaacgtgc tgcacgttcc gagtcctatt acaatggtat ttctactaac
     2461 ctgtaaggat ccactagtcc agtgtggtgg aattctgcag atatccagca cagtggcggc
     2521 cgctcgagtc tagaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2581 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2641 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2701 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2761 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2821 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2881 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2941 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg catcccttta gggttccgat
     3001 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3061 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3121 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3181 tataagggat tttggggatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3241 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3301 cccaggcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     3361 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3421 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3481 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     3541 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3601 tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgatgaaa aagcctgaac
     3661 tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga
     3721 tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat
     3781 atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc
     3841 actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga
     3901 gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa
     3961 ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg
     4021 atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta
     4081 catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga
     4141 tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg
     4201 aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga
     4261 cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc
     4321 aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga
     4381 cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata
     4441 tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg
     4501 cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc
     4561 gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg
     4621 ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtgctac
     4681 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4741 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4801 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4861 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4921 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4981 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5041 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5101 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5161 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5221 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5281 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5341 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5401 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5461 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5521 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
     5581 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5641 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5701 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5761 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     5821 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5881 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5941 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6001 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6061 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6121 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6181 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6241 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6301 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6361 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6421 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6481 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6541 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6601 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6661 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6721 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6781 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6841 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6901 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6961 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7021 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7081 aaataggggt tccgcgcaca tttccccgaa aaatggcgga aaccccggga atctaacccg
     7141 gctgaacgga tttagagtcc attcgatcta catgatcagg tttccggtgt ttcgtccttt
     7201 ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa gcatatgata
     7261 gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt actttctacg
     7321 tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattctaa ttatctctct
     7381 aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc cctcttcctg
     7441 cccgaccttc gcgatgtacg g
//
