LOCUS       sequence-318362-        7422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a Methanosarcina thermophila derived PylRS in mammalian 
            cells for genetic code expansion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7422)
  AUTHORS   Hu L, Qin X, Huang Y, Cao W, Wang C, Wang Y, Ling X, Chen H, Wu D, 
            Lin Y, Liu T
  TITLE     Thermophilic Pyrrolysyl-tRNA Synthetase Mutants for Enhanced 
            Mammalian Genetic Code Expansion.
  JOURNAL   ACS Synth Biol. 2020 Oct 16;9(10):2723-2736. doi: 
            10.1021/acssynbio.0c00257. Epub 2020 Sep 23.
  PUBMED    32931698
REFERENCE   2  (bases 1 to 7422)
  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/acssynbio.0c00257"; journalName: "ACS Synth Biol"; date: 
            "2020-10-16- 16"; volume: "9"; issue: "10"; pages: "2723-2736"
COMMENT     Sequence Label: pCMV-MtPylRS (human-opti)
FEATURES             Location/Qualifiers
     source          1..7422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(77..325)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(135..154)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(305..325)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     enhancer        502..881
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        882..1085
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1036..1056
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1082..1106
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1316..1334
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1316..1333
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1347..1366
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1399..1408
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2861..2878)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2867..3091
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3137..3565
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3224..3243)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3434..3455
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3574..3594)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3579..3909
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3760..3895
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3822..3841
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3958..4983
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3958..4983
                     /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    5113..5234
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5150..5169)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5204..5223
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5283..5299)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5283..5299)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5296..5318)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5307..5323
                     /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(5331..5337,5338..5355,5356..5361))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5376..5397
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5514..5531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5685..6273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5765..5784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6444..7235,7236..7304))
                     /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     7067..7086
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7305..7409)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaatggcgg aaaccccggg aatctaaccc ggctgaacgg atttagagtc cattcgatct
       61 acatgatcag gtttccggtg tttcgtcctt tccacaagat atataaagcc aagaaatcga
      121 aatactttca agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg
      181 caaactaccc aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt
      241 ttacagtcaa attaattcta attatctctc taacagcctt gtatcgtata tgcaaatatg
      301 aaggaatcat gggaaatagg ccctcttcct gcccgacctt cgcgatgtac gggccagata
      361 tacgccaata ttggccatta gccatattat tcattggtta tatagcataa atcaatattg
      421 gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat attggctcat
      481 gtccaatatg accgccatgt tggcattgat tattgactag ttattaatag taatcaatta
      541 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      601 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      661 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      721 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
      781 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
      841 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      901 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      961 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
     1021 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1081 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
     1141 atagaagaca ccgggaccga tccagcctcc gcggccggga acggtgcatt ggaacgcgga
     1201 ttccccgtgc caagagtgac gtaagtaccg cctatagagt ctataggccc acccccttgg
     1261 cttcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacgattta
     1321 ggtgacacta tagaataaca tccactttgc ctttctctcc acaggtgtcc actcccaggt
     1381 ccaactgcac ggaagcttgc caccatggac aagaagcctc tgaacaccct gatctctgcc
     1441 acaggcctgt ggatgagcag gaccggcaag ctgcacaaga tccgccacca cgaggtgtcc
     1501 aagcggaaga tctacatcga gatggagtgc ggcgagcgcc tggtggtgaa caattctcgc
     1561 agctgtcggg cagcaagagc cctgaggcac cacaagtata gaaagatctg caagcactgt
     1621 agggtgtccg acgaggatct gaacaagttc ctgaccagga caaacgagga taagagcaat
     1681 gccaaggtga ccgtggtgtc cgcccccaag atcagaaaag tgatgcctaa gagcgtggcc
     1741 aggacaccca agcctctgga gaataccgcc cctgtgcaga cactgccatc cgagtctcag
     1801 ccagccccca ccacaccaat cagcgcctcc accacagcac cagcatctac cagcaccaca
     1861 gcacctgccc cagcctctac cacagcacca gcacctgcaa gcaccacagc accagcctcc
     1921 gcctctacca caatcagcac ctccgccatg cctgcctcta caagcgccca gggcaccaca
     1981 aagttcaatt acatctctgg cggctttcca aggccaatcc ctgtgcaggc atccgccccc
     2041 gccctgacca agtctcagat cgacagactc cagggcctgc tgagccctaa ggacgagatc
     2101 tccctggatt ctggcacacc attcaggaag ctggagagcg agctgctgtc ccggagaagg
     2161 aaggacctga agcagatcta cgccgaggag cgcgagcact atctgggcaa gctggagcgg
     2221 gagatcacca agttctttgt ggatagaggc tttctggaga tcaagagccc catcctgatc
     2281 cctatggagt acatcgagcg catgggcatc gacaacgata aggagctgtc caagcagatc
     2341 ttccgcgtgg ataacaattt ttgcctgcgg ccaatgctgg cccccaacct gtacaattat
     2401 ctgagaaagc tgaatagggc cctgccagac cccatcaaga tctttgagat cggcccatgt
     2461 tatcggaagg agtccgatgg caaggagcac ctggaggagt tcaccatgct gaacttttgc
     2521 cagatgggct ctggctgtac aagagagaat ctggaggcca tcatcaagga cttcctggat
     2581 tacctgggca tcgactttga gatcgtgggc gattcctgca tggtgtatgg cgacaccctg
     2641 gatgtgatgc acggcgacct ggagctgagc tccgccgtgg tgggacctgt gccaatggac
     2701 agggattggg gcatcaacaa gccctggatc ggagcaggat tcggactgga gcggctgctg
     2761 aaagtgatgc acaactttaa gaatatcaag cgcgcctccc ggtctgagag ctactataac
     2821 ggcatcagca caaatctgtg aggatccacc cgctgatcag cctcgactgt gccttctagt
     2881 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2941 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3001 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     3061 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     3121 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3181 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3241 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg gggcatccct
     3301 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3361 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3421 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3481 tattcttttg atttataagg gattttgggg atttcggcct attggttaaa aaatgagctg
     3541 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     3601 agtccccagg ctccccaggc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3661 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3721 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3781 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3841 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3901 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgatg
     3961 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
     4021 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
     4081 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
     4141 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
     4201 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
     4261 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
     4321 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
     4381 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
     4441 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg
     4501 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc
     4561 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg
     4621 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt
     4681 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg
     4741 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc
     4801 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc
     4861 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt
     4921 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa
     4981 tagcacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5041 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     5101 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5161 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5221 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     5281 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5341 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5401 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5461 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5521 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5581 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5641 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5701 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5761 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5821 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     5881 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5941 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6001 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6061 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6121 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6181 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6241 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6301 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6361 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6421 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6481 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6541 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6601 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6661 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6721 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6781 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6841 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6901 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6961 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7021 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7081 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7141 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7201 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7261 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7321 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7381 tagaaaaata aacaaatagg ggttccgcgc acatttcccc ga
//
