LOCUS       sequence-325767-        7235 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a Methanosarcina flavescens 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 7235)
  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 7235)
  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-MfPylRS (human-opti)
FEATURES             Location/Qualifiers
     source          1..7235
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     complement(2174..2191)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2180..2404
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2450..2878
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2537..2556)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2747..2768
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2887..2907)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2892..3221
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3072..3207
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3134..3153
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3270..4295
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3270..4295
                     /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    4425..4546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4462..4481)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4516..4535
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4595..4611)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4595..4611)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4608..4630)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4619..4635
                     /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(4643..4649,4650..4667,4668..4673))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4688..4709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4826..4843)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4997..5585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5077..5096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5756..6547,6548..6616))
                     /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     6379..6398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6617..6721)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(6811..7059)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(6869..6888)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7039..7059)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacg gaagcttgcc
      901 atggacaaga agcccctgga tgtgctgatc tctgccaccg gactgtggat gagcaggacc
      961 ggcacactgc acaagatcaa gcaccacgag gtgtctaagc ggaagatcta catcgagatg
     1021 gcctgcggcg acagactggt ggtgaacaat agcaggaact gtcgcaccgc ccgggccttc
     1081 agacaccaca agtaccgcaa gacatgcaag tattgtaagg tgtccgacga ggatatctct
     1141 aattttctga ccaagagcac agagaacaat agctccgtga aggtgaaggt ggtgtccgcc
     1201 cccaaggtga agaaggccat gcctaagtct gtgagcaggg caccaaagcc tctggagacc
     1261 cctgtgagcg cccagacatc cgcctctcgc agcggatccg tgcacgcaaa gtctgccagc
     1321 aactctagct cctctacctc cgtgccagca cccgccctga caaggtctca gctggacagg
     1381 gtggaggccc tgctgagccc agaggacaag gtgtccctgg atgcagcaaa gcccttccgg
     1441 gagctggagt ccgagctgct ggtgcggaga aagggcgatc tgcagcggat ctacgccaac
     1501 gagagagaga attatctggg caagctggag agggacatca ccaagttctt tgtggatcgc
     1561 ggctttctgg agatcaagag cccaatcctg atccccgccg agtacgtgga gcggatgggc
     1621 atcgactccg attctgagct gtccaagcag gtgttcaggg tggacaagaa tttttgcctg
     1681 cgccctatgc tggccccaaa cctgtacaat tatctgcgga agctggacag agtgctgcct
     1741 gatccagtga agatcttcga gatcggccct tgttatagaa aggagtccga gggcaaggag
     1801 cacctggagg agttcaccat gctgaacttt tgccagatgg gctctggctg tacacgggag
     1861 aatctggagg ccctgatcag agagttcctg gactacctgg gcatcgattt tgagatcgtg
     1921 ggcgacagct gcatggtgta tggcgacacc ctggatgtga tgcacggcaa cctggagctg
     1981 agctccgccg tgatcggacc aatccctctg gatagggagt ggggcatcaa taagccatgg
     2041 atcggagcag gattcggact ggagcggctg ctgaaagtga tgcacggctt taagaacatc
     2101 aagagggccg cccgcagcga gtcctactat aatggcatct ctacaagcct gtgaggatcc
     2161 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2221 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2281 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2341 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2401 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     2461 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2521 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2581 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2641 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2701 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2761 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2821 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     2881 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2941 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3001 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3061 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3121 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     3181 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     3241 tatccatttt cggatctgat cagcacgtga tgaaaaagcc tgaactcacc gcgacgtctg
     3301 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     3361 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     3421 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     3481 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     3541 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     3601 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     3661 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     3721 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     3781 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     3841 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     3901 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     3961 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     4021 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     4081 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     4141 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     4201 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     4261 gacgccccag cactcgtccg agggcaaagg aatagcacgt gctacgagat ttcgattcca
     4321 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4381 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4441 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4501 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4561 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4621 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4681 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4741 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4801 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4861 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4921 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4981 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5041 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5101 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5161 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5221 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5281 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5341 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5401 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5461 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5521 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5581 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5641 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5701 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5761 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5821 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5881 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5941 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6001 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6061 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6121 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6181 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6241 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6301 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6361 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6421 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6481 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6541 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6601 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6661 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6721 gcacatttcc ccgaaaaatg gcggaaaccc cgggaatcta acccggctga acggatttag
     6781 agtccattcg atctacatga tcaggtttcc ggtgtttcgt cctttccaca agatatataa
     6841 agccaagaaa tcgaaatact ttcaagttac ggtaagcata tgatagtcca ttttaaaaca
     6901 taattttaaa actgcaaact acccaagaaa ttattacttt ctacgtcacg tattttgtac
     6961 taatatcttt gtgtttacag tcaaattaat tctaattatc tctctaacag ccttgtatcg
     7021 tatatgcaaa tatgaaggaa tcatgggaaa taggccctct tcctgcccga ccttcgcgat
     7081 gtacgggcca gatatacgcc aatattggcc attagccata ttattcattg gttatatagc
     7141 ataaatcaat attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat
     7201 ttatattggc tcatgtccaa tatgaccgcc atgtt
//
