LOCUS       sequence-325763-        7421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a Methanosarcina thermophila derived AcKRS in mammalian 
            cells for genetic code expansion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7421)
  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 7421)
  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-MtAcKRS (human-opti)
FEATURES             Location/Qualifiers
     source          1..7421
                     /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"
     regulatory      898..907
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2360..2377)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2366..2590
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2636..3064
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2723..2742)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2933..2954
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3073..3093)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3078..3407
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3258..3393
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3320..3339
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3456..4481
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3456..4481
                     /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    4611..4732
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4648..4667)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4702..4721
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4781..4797)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4781..4797)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4794..4816)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4805..4821
                     /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(4829..4835,4836..4853,4854..4859))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4874..4895
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5012..5029)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5183..5771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5263..5282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5942..6733,6734..6802))
                     /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     6565..6584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6803..6907)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(6997..7245)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7055..7074)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7225..7245)
                     /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 accatggaca agaagcctct gaacaccctg atctctgcca caggcctgtg gatgagcagg
      961 accggcaagc tgcacaagat ccgccaccac gaggtgtcca agcggaagat ctacatcgag
     1021 atggagtgcg gcgagcgcct ggtggtgaac aattctcgca gctgtcgggc agcaagagcc
     1081 ctgaggcacc acaagtatag aaagatctgc aagcactgta gggtgtccgg cgaggatctg
     1141 aacaagttcc tgaccaggac aaacgaggat aagagcaatg ccaaggtgac cgtggtgtcc
     1201 gcccccaaga tcagaaaagt gatgcctaag agcgtggcca ggacacccaa gcctctggag
     1261 aataccgccc ctgtgcagac actgccatcc gagtctcagc cagcccccac cacaccaatc
     1321 agcgcctcca ccacagcacc agcatctacc agcaccacag cacctgcccc agcctctacc
     1381 acagcaccag cacctgcaag caccacagca ccagcctccg cctctaccac aatcagcacc
     1441 tccgccatgc ctgcctctac aagcgcccag ggcaccacaa agttcaatta catctctggc
     1501 ggctttccaa ggccaatccc tgtgcaggca tccgcccccg ccctgaccaa gtctcagatc
     1561 gacagactcc agggcctgct gagccctaag gacgagatct ccctggattc tggcacacca
     1621 ttcaggaagc tggagagcga gctgctgtcc cggagaagga aggacctgaa gcagatctac
     1681 gccgaggagc gcgagcacta tctgggcaag ctggagcggg agatcaccaa gttctttgtg
     1741 gatagaggct ttctggagat caagagcccc atcctgatcc ctatggagta catcgagcgc
     1801 atgggcatcg acaacgataa ggagctgtcc aagcagatct tccgcgtgga taacaatttt
     1861 tgcctgcggc caatgatggc ccccaacatc ttcaattatg ccagaaagct gaatagggcc
     1921 ctgccagacc ccatcaagat ctttgagatc ggcccatgtt atcggaagga gtccgatggc
     1981 aaggagcacc tggaggagtt caccatgctg aactttttcc agatgggctc tggctgtaca
     2041 agagagaatc tggaggccat catcaaggac ttcctggatt acctgggcat cgactttgag
     2101 atcgtgggcg attcctgcat ggtgtatggc gacaccctgg atgtgatgca cggcgacctg
     2161 gagctgagct ccgccgtggt gggacctgtg ccaatggaca gggattgggg catcaacaag
     2221 ccctggatcg gagcaggatt cggactggag cggctgctga aagtgatgca caactttaag
     2281 aatatcaagc gcgcctcccg gtctgagagc tactataacg gcatcagcac aaatctgtga
     2341 ggatccaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2401 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2461 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2521 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2581 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2641 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2701 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2761 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2821 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2881 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2941 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3001 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3061 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3121 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3181 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3241 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3301 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3361 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3421 cttgtatatc cattttcgga tctgatcagc acgtgatgaa aaagcctgaa ctcaccgcga
     3481 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
     3541 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     3601 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     3661 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct
     3721 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
     3781 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
     3841 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     3901 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     3961 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     4021 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     4081 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     4141 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     4201 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     4261 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     4321 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     4381 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     4441 gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtgcta cgagatttcg
     4501 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4561 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4621 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4681 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4741 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4801 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4861 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4921 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4981 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5041 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5101 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5161 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5221 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5281 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5341 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5401 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5461 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5521 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5581 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5641 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5701 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5761 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5821 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5881 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5941 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6001 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6061 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6121 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6181 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6241 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6301 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6361 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6421 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6481 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6541 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6601 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6661 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6721 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6781 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6841 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6901 ttccgcgcac atttccccga aaaatggcgg aaaccccggg aatctaaccc ggctgaacgg
     6961 atttagagtc cattcgatct acatgatcag gtttccggtg tttcgtcctt tccacaagat
     7021 atataaagcc aagaaatcga aatactttca agttacggta agcatatgat agtccatttt
     7081 aaaacataat tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt
     7141 ttgtactaat atctttgtgt ttacagtcaa attaattcta attatctctc taacagcctt
     7201 gtatcgtata tgcaaatatg aaggaatcat gggaaatagg ccctcttcct gcccgacctt
     7261 cgcgatgtac gggccagata tacgccaata ttggccatta gccatattat tcattggtta
     7321 tatagcataa atcaatattg gctattggcc attgcatacg ttgtatctat atcataatat
     7381 gtacatttat attggctcat gtccaatatg accgccatgt t
//
