LOCUS       sequence-240743-        8196 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for PURE system components.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8196)
  AUTHORS   Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, 
            Ueda T
  TITLE     Cell-free translation reconstituted with purified components.
  JOURNAL   Nat Biotechnol. 2001 Aug;19(8):751-5. doi: 10.1038/90802.
  PUBMED    11479568
REFERENCE   2  (bases 1 to 8196)
  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"; journalName: 
            "Nat Biotechnol"; date: "2001-08"; volume: "19"; issue: "8"; pages: 
            "751-5"
COMMENT     Sequence Label: pET21a-ValRS-His
FEATURES             Location/Qualifiers
     source          1..8196
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    3041..3065
                     /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     complement(3065..3084)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3066..3084)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3168..3187)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3393..3470
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3471..4553
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3490..3509)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5362..5553
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5547..5569)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5655..5794
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5710..5729
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5809..5826)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5980..6568)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6060..6079)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6739..7530,7531..7599))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7362..7381
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7600..7704)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7730..8185)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7867..7888)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8079..8098
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcggcgataa
      181 cagcctgctg ttcaatcagt ttcgctttcg cttccgcata gccttccagc ttctcacgct
      241 ctttcgcgat gaccgcttcc ggtgcgcggg cgacaaagcc ttcgttcgcc agtttgttct
      301 cgatacggct gatttcacct tcaatcttcg ccacttcttt cgccagacgc gccagctcat
      361 cttctttgtt gatgaggcca gccatcggga tcagcagctc tgcaccgtcg atgatcttcg
      421 taacggaaac cggacctttg tcatcggcag gcagcacggt gatactttcc agacgcgcca
      481 gggtttgcag gaagccacgg ttttcattta cgcgacgttc tgcatccgcg ctgcaaccac
      541 gcagcagcag ctccagcggt ttgcccggcg cgatgttcat ttctgcacgg atgttacgta
      601 ccgcaacgat cgcctgtttc agccattcgg tgtcggccag tgcggcttca tcaacctgag
      661 atgcatcgta ctgcgggaac ggctgcagca tgatggtgtc ggcagtgata ccgcaaagta
      721 ctttcacacg ctgccagatg gtttcggtga tgaacggaat gatcggatgc gcgaggcgca
      781 gcagaccttc cagtacagtc accagcgtat ggcgagtacc gcgcagttct gcttcggtgc
      841 caccgttcat taccggcttg gtcagctcga gataccagtc acagaactgg ttccaggtga
      901 actcatacag aatgcctgcg gcgatatcga agcggaagct gtccagcgct tcgcggtacg
      961 ctttgatggt ctggttgaac tccgccagaa tccagcggtc cgccagcgac agcgtcattt
     1021 cgccgccgtt gaagccgcaa tcctgacctt ctgtgttcat cagcacaaag cggctggcgt
     1081 tccacagctt gttacagaag ttacggtaac cttccagacg cttcatatcc cagttgatgt
     1141 cacgaccggt agacgccagc gccgccaggg tgaagcgcag cgcgtcagta ccgtgcggct
     1201 caataccgtt cgggaactgc ttctcggtgc gcttacggat tttgtccgcc agctgcggct
     1261 gcatcatatt gccggtacgt ttttccagca gttctggcag cgaaataccg tcaaccatat
     1321 ccagtgggtc gataacgtta cccttggatt tggacatctt ctggccttcg tcatcacgaa
     1381 tcaggccggt catgtaaacg gtgtggaacg gcacctgcgg tttgccattt tcatctttga
     1441 tgaagtgcat ggtcatcatg atcatgcggg caatccagaa gaaaatgatg tcgaaaccag
     1501 ataccatcac gctggttggg tggaactgac gcagggcgtc ggtattttcc ggccagccaa
     1561 gggtagagaa ggtccacagc gcagaagaga accaggtatc gagaacgtct tcgtcctgac
     1621 gcaggacaac atcagcaccg aggttatttt ctttacgcac ttcgtcttcg ttgcggccaa
     1681 cataaacgtt acccgcttcg tcataccatg ccgggatacg gtgaccccac cacaactgac
     1741 gagagataca ccagtcctga atatcgcgca tccaggagaa gtacatgttt tcgtactgct
     1801 tcggtacgaa ctgaatgtcg ccgttctcaa ccgcttcaac cgccggtttc gccaggacat
     1861 cggcacgcac gtaccactgg tcggtcagca ttggttcgat aactacgccg ccacggtcgc
     1921 cgtaaggaac ggtcaggtcg tgcggtttaa tttcttccag caggccaagc gcgtcaactg
     1981 cggcaacgac tgctttacgt gcagcaaaac gctccagttt ctggaactct gcagggattt
     2041 cgctggaata aacgtcagat tcgttacctt tggtatcgaa cacctgggcg ctttcacgga
     2101 tatcgccgtc aaaggtcagg atgttgatca tcggcagggc gtgacgttta cccacttcat
     2161 agtcgttaaa gtcgtgcgcc ggagtgattt tcacgcagcc ggtgcctttt tccatgtcgg
     2221 cgtgttcgtc gccaacgatc ggaatacgac ggttaaccag cggcagaatg acatatttgc
     2281 caatcagatc tttgtaacgc ggatcttccg ggttaacggc tacgccagta tcgcccagca
     2341 gggtttctgg acgggtagtc gcgaccacca gataatcttt accgtctgcg gttttcgcac
     2401 cgtcagccag cggatagcgg atgtgccaca tcgaaccttt cgattcgcgg ttttcgactt
     2461 ccaggtcaga gatagcggtg cgcagtttcg gatcccagtt taccaggcgt ttgccacggt
     2521 aaatcaggtc ttctttatac agacgaacga aaacttcttt caccgcattg gacaggcctt
     2581 cgtccatggt gaagcgttca cgctcccagt cgacggagtt gccgagacgg cgcatctgac
     2641 gggtaatggt gccgccagat tccgctttcc attcccagat tttgtcgatg aaagcttcgc
     2701 ggccgtagtc gtgacgggtt ttaccttctt ctgcggcaat cttgcgctca acgaccatct
     2761 gggtagcgat cccggcgtgg tcagtaccga cctgccacag ggtgtttttg ccctgcatgc
     2821 gctgatagcg gatcatggta tccatgatgg tttgctggaa ggcgtgaccc atatgcaaac
     2881 tgccggtgac gttcggcggc gggatcatga tgcagaaact ttcctggctt tcatcgccat
     2941 taggcttaaa gtagccctgc ttttcccagt gctcgtaaag cggctgttcg atatcttgtg
     3001 ggttatatgt cttttccatt tttccatctc cttctagagg ggaattgtta tccgctcaca
     3061 attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc tctacgccgg
     3121 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     3181 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     3241 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
     3301 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
     3361 ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc
     3421 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     3481 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     3541 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     3601 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     3661 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     3721 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     3781 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     3841 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     3901 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     3961 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     4021 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
     4081 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
     4141 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg
     4201 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg
     4261 ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc
     4321 cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct
     4381 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg
     4441 tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     4501 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     4561 gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga
     4621 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     4681 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt
     4741 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc
     4801 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc
     4861 gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg
     4921 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg
     4981 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca
     5041 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc
     5101 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca
     5161 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc
     5221 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta
     5281 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat
     5341 cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc
     5401 tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa
     5461 caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc
     5521 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     5581 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     5641 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc
     5701 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc
     5761 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     5821 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5881 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5941 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6001 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6061 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6121 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6181 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6241 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6301 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6361 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6421 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6481 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6541 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6601 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6661 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6721 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6781 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6841 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6901 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6961 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7021 agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc
     7081 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7141 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7201 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7261 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7321 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7381 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7441 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7501 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7561 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7621 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7681 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     7741 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     7801 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     7861 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     7921 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     7981 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     8041 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     8101 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     8161 cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
