LOCUS       sequence-445174-        7460 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 7460)
  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 7460)
  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..7460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(55..74)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(87..105)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(88..105)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(315..339)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(336..539)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(365..385)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        540..919
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1096..1344
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1096..1116
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1267..1286
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        1434..1538
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1539..1607,1608..2399)
                     /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(1757..1776)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2570..3158
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3059..3078
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3312..3329
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3446..3467
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3482..3487,3488..3505,3506..3512)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3520..3536
                     /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     3525..3547
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3544..3560
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3544..3560
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3609..3730
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3620..3639)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3674..3693
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3860..4885)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(3860..4885)
                     /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"
     promoter        complement(4934..5263)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4948..5083
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5002..5021)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5248..5268
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5277..5705)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5387..5408)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5599..5618
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5751..5975
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5964..5981
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(7428..7454)
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
ORIGIN
        1 cggcaccggg cacgccatgg tgaagcttcc gtgcagttgg acctgggagt ggacacctgt
       61 ggagagaaag gcaaagtgga tgttattcta tagtgtcacc taaatcgtat gtgtatgata
      121 cataaggtta tgtattaatt gtagccgcgt tctaacgaag ccaagggggt gggcctatag
      181 actctatagg cggtacttac gtcactcttg gcacggggaa tccgcgttcc aatgcaccgt
      241 tcccggccgc ggaggctgga tcggtcccgg tgtcttctat ggaggtcaaa acagcgtgga
      301 tggcgtctcc aggcgatctg acggttcact aaacgagctc tgcttatata gacctcccac
      361 cgtacacgcc taccgcccat ttgcgtcaac ggggcggggt tattacgaca ttttggaaag
      421 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
      481 aatccccgtg agtcaaaccg ctatccacgc ccattggtgt actgccaaaa ccgcatcacc
      541 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccgtaaggtc
      601 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgga
      661 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
      721 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
      781 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
      841 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
      901 ctagtcaata atcaatgcca acatggcggt catattggac atgagccaat ataaatgtac
      961 atattatgat atagatacaa cgtatgcaat ggccaatagc caatattgat ttatgctata
     1021 taaccaatga ataatatggc taatggccaa tattggcgta tatctggccc gtacatcgcg
     1081 aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac
     1141 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
     1201 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     1261 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     1321 atcttgtgga aaggacgaaa caccggaaac ctgatcatgt agatcgaatg gactctaaat
     1381 ccgttcagcc gggttagatt cccggggttt ccgccatttt tcggggaaat gtgcgcggaa
     1441 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1501 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1561 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1621 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1681 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1741 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1801 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1861 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1921 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1981 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     2041 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     2101 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     2161 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     2221 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     2281 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2341 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2401 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2461 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2521 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2581 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2641 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2701 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2761 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2821 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2881 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2941 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3001 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3061 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3121 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3181 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3241 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3301 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     3361 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     3421 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     3481 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     3541 acacaggaaa cagctatgac catgattacg ccaagctcta gctagaggtc gacggtatac
     3601 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3661 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3721 taaacaagtt ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc
     3781 agccggcgtc ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat
     3841 cgaaatctcg tagcacgtgc tattcctttg ccctcggacg agtgctgggg cgtcggtttc
     3901 cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt ctgcgggcga
     3961 tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat cgaccctgcg
     4021 cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg tcaagaccaa
     4081 tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc ctccgctcga
     4141 agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag atgttggcga
     4201 cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct gttatgcggc
     4261 cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc cggacttcgg
     4321 ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac gcactgacgg
     4381 tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg catatgaaat
     4441 cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac ccgctcgtct
     4501 ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc agaacagcgg
     4561 gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg gagatgcaat
     4621 aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg agcgcggccg
     4681 atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag ctatttaccc
     4741 gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct tcgccctccg
     4801 agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc tcgacagacg
     4861 tcgcggtgag ttcaggcttt ttcatcacgt gctgatcaga tccgaaaatg gatatacaag
     4921 ctcccgggag ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga
     4981 atagctcaga ggcagaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat
     5041 ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg
     5101 ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     5161 ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc
     5221 ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac agaattaatt
     5281 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     5341 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     5401 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     5461 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     5521 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     5581 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     5641 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     5701 cgcgtgggga taccccctag agccccagct ggttctttcc gcctcagaag ccatagagcc
     5761 caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc
     5821 caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt
     5881 ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg
     5941 gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gggttctaga
     6001 ctcgagcggc cgccactgtg ctggatatct gcagaattcc accacactgg actagtggat
     6061 ccttacaggt tagtagaaat accattgtaa taggactcgg aacgtgcagc acgtttgatg
     6121 ttcttgaagt cgtgttttac tttcagcaga cgctccagac caaaacccgc accgatccaa
     6181 ggtttgtcga taccccactc acgatccagc gggattgggc ccacaacggc actagacagt
     6241 tccaggtcgc cgtgcatgac atccagggtg tcgccaaaca ccatacagct gtcgcccaca
     6301 attttgaagt caatgcccag gtggttcaga aaatcggtga tgatgctttc caggttctca
     6361 cgagtacaac ctgaacccat ttggcaaaag ttcagcatgg taaactcctc cagatgttct
     6421 ttaccgtcgg actctttacg ataacacggg ccgatctcga agattttgat aggatcaggc
     6481 agggcacggt ccagtttgcg cagatagtta gccagatttg gtgctagcat agggcgcaga
     6541 cagaagtttt tatccacacg gaaaatttgt ttgctcagtt cggtatcatt gtcgatgccc
     6601 atacgctcga tatactccag aggaatcaga atcggggatt tgatctccag aaagccacga
     6661 tccacgaaaa agcgggtgat ttcacgttcc agtttcccca gatagttctc acgttcttcg
     6721 gcatagattt gttgcaggtc ttttttacga cgtgacagca gttcgctctc cagttcacga
     6781 aacggtttgc cggaattcag gctgatttcg tctttcggat tcagcagaac ctccagacga
     6841 tcggtttggg attttgtcag tgctggagct gatgcttgaa ccggggcaga catgcttgta
     6901 atcggattgg tattgccttt aaccagggcg ctagcggtgg caccggtgct aatactgcta
     6961 atgctggtgc tcacacttgc tggaacagaa acggactcct gggtagaaac aggaatggcc
     7021 ggagagaatt tgcttccaga cggctgtgcc tgtgctgctt cagtgttttc cagtggttta
     7081 ggggcacgag caacggattt cggcattgct tttttagtac gggtaggagc gctaacgact
     7141 ttcactttca cgcttgtttg gtcctcattg gcttttgtca ggaatttgtt cagatcctca
     7201 tcggacacac ggcaacgttt acaggtttta cgatatttgt ggtgacgcag tgcacgtgct
     7261 gtacgagaag agcggctatt gttcacaacc agatgatcgc cacacgccat ctcaatatag
     7321 attttcgaac ggctaacctc gtggtgtttg attttatgaa tggttccggt acgactcatc
     7381 cacagaccag tagcagagat cagggtattc agcggttttt tatcatccag ggtcaggcgt
     7441 tccagcggcg gcagctgcag
//
