LOCUS       sequence-325765-        7238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a Methanosarcina flavescens derived BulKRS in mammalian 
            cells for genetic code expansion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7238)
  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 7238)
  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-MfBulKRS (human-opti)
FEATURES             Location/Qualifiers
     source          1..7238
                     /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(2177..2194)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2183..2407
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2453..2881
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2540..2559)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2750..2771
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2890..2910)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2895..3224
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3075..3210
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3137..3156
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3273..4298
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3273..4298
                     /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    4428..4549
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4465..4484)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4519..4538
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4598..4614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4598..4614)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4611..4633)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4622..4638
                     /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(4646..4652,4653..4670,4671..4676))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4691..4712
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4829..4846)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5000..5588)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5080..5099)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5759..6550,6551..6619))
                     /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     6382..6401
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6620..6724)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(6814..7062)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(6872..6891)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7042..7062)
                     /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 agaagcccct ggatgtgctg atctctgcca ccggactgtg gatgagcagg
      961 accggcacac tgcacaagat caagcaccac gaggtgtcta agcggaagat ctacatcgag
     1021 atggcctgcg gcgacagact ggtggtgaac aatagcagga actgtcgcac cgcccgggcc
     1081 ttcagacacc acaagtaccg caagacatgc aagtattgta aggtgtccga cgaggatatc
     1141 tctaattttc tgaccaagag cacagagaac aatagctccg tgaaggtgaa ggtggtgtcc
     1201 gcccccaagg tgaagaaggc catgcctaag tctgtgagca gggcaccaaa gcctctggag
     1261 acccctgtga gcgcccagac atccgcctct cgcagcggat ccgtgcacgc aaagtctgcc
     1321 agcaactcta gctcctctac ctccgtgcca gcacccgccc tgacaaggtc tcagctggac
     1381 agggtggagg ccctgctgag cccagaggac aaggtgtccc tggatgcagc aaagcccttc
     1441 cgggagctgg agtccgagct gctggtgcgg agaaagggcg atctgcagcg gatctacgcc
     1501 aacgagagag agaattatct gggcaagctg gagagggaca tcaccaagtt ctttgtggat
     1561 cgcggctttc tggagatcaa gagcccaatc ctgatccccg ccgagtacgt ggagcggatg
     1621 ggcatcgact ccgattctga gctgtccaag caggtgttca gggtggacaa gaatttttgc
     1681 ctgcgcccta tgctggcccc aaacctggcc aattatctgc ggaagctgga cagagtgctg
     1741 cctgatccag tgaagatctt cgagatcggc ccttgttata gaaaggagtc cgagggcaag
     1801 gagcacctgg aggagttcac catgctgaac ttttgccaga tgggctctgg ctgtacacgg
     1861 gagaatctgg aggccctgat cagagagttc ctggactacc tgggcatcga ttttgagatc
     1921 gtgggcgaca gctgcatggt gttcggcgac accctggatg tgatgcacgg caacctggag
     1981 ctgagctccg ccgtgatcgg accaatccct ctggataggg agtggggcat caataagcca
     2041 tggatcggag caggattcgg actggagcgg ctgctgaaag tgatgcacgg ctttaagaac
     2101 atcaagaggg ccgcccgcag cgagtcctac tataatggca tctctacaag cctgtgagga
     2161 tccacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     2221 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     2281 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     2341 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     2401 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     2461 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2521 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2581 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2641 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2701 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2761 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2821 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2881 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2941 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3001 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3061 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3121 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     3181 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     3241 gtatatccat tttcggatct gatcagcacg tgatgaaaaa gcctgaactc accgcgacgt
     3301 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     3361 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     3421 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     3481 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     3541 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     3601 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     3661 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     3721 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     3781 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     3841 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     3901 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     3961 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     4021 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     4081 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     4141 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     4201 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     4261 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtgctacga gatttcgatt
     4321 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4381 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4441 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4501 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4561 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4621 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4681 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4741 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4801 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4861 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4921 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4981 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5041 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5101 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5161 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5221 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5281 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5341 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5401 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5461 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5521 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5581 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5641 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5701 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5761 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5821 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5881 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5941 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6001 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6061 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6121 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6181 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6241 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6301 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6361 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6421 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6481 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6541 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6601 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6661 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6721 cgcgcacatt tccccgaaaa atggcggaaa ccccgggaat ctaacccggc tgaacggatt
     6781 tagagtccat tcgatctaca tgatcaggtt tccggtgttt cgtcctttcc acaagatata
     6841 taaagccaag aaatcgaaat actttcaagt tacggtaagc atatgatagt ccattttaaa
     6901 acataatttt aaaactgcaa actacccaag aaattattac tttctacgtc acgtattttg
     6961 tactaatatc tttgtgttta cagtcaaatt aattctaatt atctctctaa cagccttgta
     7021 tcgtatatgc aaatatgaag gaatcatggg aaataggccc tcttcctgcc cgaccttcgc
     7081 gatgtacggg ccagatatac gccaatattg gccattagcc atattattca ttggttatat
     7141 agcataaatc aatattggct attggccatt gcatacgttg tatctatatc ataatatgta
     7201 catttatatt ggctcatgtc caatatgacc gccatgtt
//
