LOCUS       sequence-448627-        7392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PylRS (WT)/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 7392)
  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 7392)
  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(WT)_hU6tRNAPyl
FEATURES             Location/Qualifiers
     source          1..7392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(321..650)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      335..470
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(389..408)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     635..655
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(664..1092)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(774..795)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     986..1005
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1138..1362
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1351..1368
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(2747..2773)
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(2834..2853)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(2866..2884)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2867..2884)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3094..3118)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(3115..3318)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3144..3164)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3319..3698
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3875..4123
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3875..3895
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4046..4065
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        4213..4317
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4318..4386,4387..5178)
                     /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     complement(4536..4555)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5349..5937
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5838..5857
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6091..6108
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6225..6246
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6261..6266,6267..6284,6285..6291)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6299..6315
                     /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)."
     primer_bind     6304..6326
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6323..6339
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6323..6339
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    6388..6509
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6399..6418)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6453..6472
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(6639..7392,1..272))
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(join(6639..7392,1..272))
                     /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"
ORIGIN
        1 atgcaatagg tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc
       61 gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc ggcgcagcta
      121 tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg
      181 ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag aaacttctcg
      241 acagacgtcg cggtgagttc aggctttttc atcacgtgct gatcagatcc gaaaatggat
      301 atacaagctc ccgggagctt tttgcaaaag cctaggcctc caaaaaagcc tcctcactac
      361 ttctggaata gctcagaggc agaggcggcc tcggcctctg cataaataaa aaaaattagt
      421 cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt
      481 aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
      541 ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata
      601 cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac attccacaga
      661 attaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
      721 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
      781 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
      841 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
      901 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
      961 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     1021 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     1081 tacagggcgc gtggggatac cccctagagc cccagctggt tctttccgcc tcagaagcca
     1141 tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt
     1201 cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt
     1261 cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg
     1321 gggaggggca aacaacagat ggctggcaac tagaaggcac agtcgaggct gatcagcggg
     1381 tttacaggtt agtagaaata ccattgtaat aggactcgga acgtgcagca cgtttgatgt
     1441 tcttgaagtc gtgttttact ttcagcagac gctccagacc aaaacccgca ccgatccaag
     1501 gtttgtcgat accccactca cgatccagcg ggattgggcc cacaacggca ctagacagtt
     1561 ccaggtcgcc gtgcatgaca tccagggtgt cgccatacac catacagctg tcgcccacaa
     1621 ttttgaagtc aatgcccagg tggttcagaa aatcggtgat gatgctttcc aggttctcac
     1681 gagtacaacc tgaacccatt tggcaaaagt tcagcatggt aaactcctcc agatgttctt
     1741 taccgtcgga ctctttacga taacacgggc cgatctcgaa gattttgata ggatcaggca
     1801 gggcacggtc cagtttgcgc agatagttat acagatttgg tgccagcata gggcgcagac
     1861 agaagttttt atccacacgg aaaatttgtt tgctcagttc ggtatcattg tcgatgccca
     1921 tacgctcgat atactccaga ggaatcagaa tcggggattt gatctccaga aagccacgat
     1981 ccacgaaaaa gcgggtgatt tcacgttcca gtttccccag atagttctca cgttcttcgg
     2041 catagatttg ttgcaggtct tttttacgac gtgacagcag ttcgctctcc agttcacgaa
     2101 acggtttgcc ggaattcagg ctgatttcgt ctttcggatt cagcagaacc tccagacgat
     2161 cggtttggga ttttgtcagt gctggagctg atgcttgaac cggggcagac atgcttgtaa
     2221 tcggattggt attgccttta accagggcgc tagcggtggc accggtgcta atactgctaa
     2281 tgctggtgct cacacttgct ggaacagaaa cggactcctg ggtagaaaca ggaatggccg
     2341 gagagaattt gcttccagac ggctgtgcct gtgctgcttc agtgttttcc agtggtttag
     2401 gggcacgagc aacggatttc ggcattgctt ttttagtacg ggtaggagcg ctaacgactt
     2461 tcactttcac gcttgtttgg tcctcattgg cttttgtcag gaatttgttc agatcctcat
     2521 cggacacacg gcaacgttta caggttttac gatatttgtg gtgacgcagt gcacgtgctg
     2581 tacgagaaga gcggctattg ttcacaacca gatgatcgcc acacgccatc tcaatataga
     2641 ttttcgaacg gctaacctcg tggtgtttga ttttatgaat ggttccggta cgactcatcc
     2701 acagaccagt agcagagatc agggtattca gcggtttttt atcatccagg gtcaggcgtt
     2761 ccagcggcgg cagctgcagc ggcaccgggc acgccatggt gaagcttccg tgcagttgga
     2821 cctgggagtg gacacctgtg gagagaaagg caaagtggat gttattctat agtgtcacct
     2881 aaatcgtatg tgtatgatac ataaggttat gtattaattg tagccgcgtt ctaacgaagc
     2941 caagggggtg ggcctataga ctctataggc ggtacttacg tcactcttgg cacggggaat
     3001 ccgcgttcca atgcaccgtt cccggccgcg gaggctggat cggtcccggt gtcttctatg
     3061 gaggtcaaaa cagcgtggat ggcgtctcca ggcgatctga cggttcacta aacgagctct
     3121 gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaacg gggcggggtt
     3181 attacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc
     3241 aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattggtgta
     3301 ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt
     3361 aggaaagtcc cgtaaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt
     3421 gacgtcaata gggggcggac ttggcatatg atacacttga tgtactgcca agtgggcagt
     3481 ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg
     3541 aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc
     3601 atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt
     3661 aattgattac tattaataac tagtcaataa tcaatgccaa catggcggtc atattggaca
     3721 tgagccaata taaatgtaca tattatgata tagatacaac gtatgcaatg gccaatagcc
     3781 aatattgatt tatgctatat aaccaatgaa taatatggct aatggccaat attggcgtat
     3841 atctggcccg tacatcgcga aggtcgggca ggaagagggc ctatttccca tgattccttc
     3901 atatttgcat atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa
     3961 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     4021 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     4081 cgatttcttg gctttatata tcttgtggaa aggacgaaac accggaaacc tgatcatgta
     4141 gatcgaatgg actctaaatc cgttcagccg ggttagattc ccggggtttc cgccattttt
     4201 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4261 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4321 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4381 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4441 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4501 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4561 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4621 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4681 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4741 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4801 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4861 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4921 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4981 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5041 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5101 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5161 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5221 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5281 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5341 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5401 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5461 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5521 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5581 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5641 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5701 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5761 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5821 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5881 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5941 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6001 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6061 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     6121 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     6181 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     6241 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     6301 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagctctag
     6361 ctagaggtcg acggtataca gacatgataa gatacattga tgagtttgga caaaccacaa
     6421 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     6481 taaccattat aagctgcaat aaacaagttg gggtgggcga agaactccag catgagatcc
     6541 ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca
     6601 tagaaggcgg cggtggaatc gaaatctcgt agcacgtgct attcctttgc cctcggacga
     6661 gtgctggggc gtcggtttcc actatcggcg agtacttcta cacagccatc ggtccagacg
     6721 gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt
     6781 gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac caagctctga
     6841 tagagttggt caagaccaat gcggagcata tacgcccgga gccgcggcga tcctgcaagc
     6901 tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc
     6961 cagaagaaga tgttggcgac ctcgtattgg gaatccccga acatcgcctc gctccagtca
     7021 atgaccgctg ttatgcggcc attgtccgtc aggacattgt tggagccgaa atccgcgtgc
     7081 acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca tcagctcatc gagagcctgc
     7141 gcgacggacg cactgacggt gtcgtccatc acagtttgcc agtgatacac atggggatca
     7201 gcaatcgcgc atatgaaatc acgccatgta gtgtattgac cgattccttg cggtccgaat
     7261 gggccgaacc cgctcgtctg gctaagatcg gccgcagcga tcgcatccat ggcctccgcg
     7321 accggctgca gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt
     7381 gcacggcggg ag
//
