LOCUS       sequence-258590-        8213 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 8213)
  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 8213)
  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-His-IleRS
FEATURES             Location/Qualifiers
     source          1..8213
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(778..1860)
                     /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     1822..1841
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1861..1938)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2144..2163
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2247..2266
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2247..2265
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2266..2290
                     /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)."
     RBS             2305..2327
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2338..2355
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5191..5208
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5261..5279)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5275..5322
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5359..5814
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5446..5465)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5656..5677
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5840..5944
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5945..6013,6014..6805)
                     /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     complement(6163..6182)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6976..7564
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7465..7484
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7718..7735
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7750..7889
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7815..7834)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7975..7997
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7991..8182)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
       61 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      121 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
      181 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
      241 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
      301 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
      361 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
      421 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
      481 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
      541 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
      601 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
      661 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
      721 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
      781 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      841 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
      901 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
      961 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1021 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     1081 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1141 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1201 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     1261 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     1321 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     1381 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     1441 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     1501 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     1561 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1621 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1681 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1741 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     1801 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     1861 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1921 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1981 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     2041 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     2101 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     2161 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     2221 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     2281 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgcac
     2341 caccaccacc accacagtga ctataaatca accctgaatt tgccggaaac agggttcccg
     2401 atgcgtggcg atctcgccaa gcgcgaaccc ggaatgctgg cgcgttggac tgatgatgat
     2461 ctgtacggca tcatccgtgc ggctaaaaaa ggcaaaaaaa ccttcattct gcatgatggc
     2521 cctccttatg cgaatggcag cattcatatt ggtcactcgg ttaacaagat tctgaaagac
     2581 attatcgtga agtccaaagg gctttccggt tatgactcgc cgtatgtgcc tggctgggac
     2641 tgccacggtc tgccgatcga gctgaaagtc gagcaagaat acggtaagcc gggtgagaaa
     2701 ttcaccgccg ccgagttccg cgccaagtgc cgcgaatacg cggcgaccca ggttgacggt
     2761 caacgcaaag actttatccg tctgggcgtg ctgggcgact ggtcgcaccc gtacctgacc
     2821 atggacttca aaactgaagc caacatcatc cgcgcgctgg gcaaaatcat cggcaacggt
     2881 cacctgcaca aaggcgcgaa gccagttcac tggtgcgttg actgccgttc tgcgctggcg
     2941 gaagcggaag ttgagtatta cgacaaaact tctccgtcca tcgacgttgc tttccaggca
     3001 gtcgatcagg atgcactgaa agcaaaattt gccgtaagca acgttaacgg cccaatctcg
     3061 ctggtaatct ggaccaccac gccgtggact ctgcctgcca accgcgcaat ctctattgca
     3121 ccagatttcg actatgcgct ggtgcagatc gacggtcagg ccgtgattct ggcgaaagat
     3181 ctggttgaaa gcgtaatgca gcgtatcggc gtgaccgatt acaccattct cggcacggta
     3241 aaaggtgcgg agcttgagct gctgcgcttt acccatccgt ttatgggctt cgacgttccg
     3301 gcaatcctcg gcgatcacgt taccctggat gccggtaccg gtgccgttca caccgcgcct
     3361 ggccacggcc cggacgacta tgtgatcggt cagaaatacg gcctggaaac cgctaacccg
     3421 gttggcccgg acggcactta tctgccgggc acttatccga cgctggatgg cgtgaacgtc
     3481 ttcaaagcga acgacatcgt cgttgcgctg ctgcaggaaa aaggcgcgct gctgcacgtt
     3541 gagaaaatgc agcacagcta tccgtgctgc tggcgtcaca aaacgccgat catcttccgc
     3601 gcgacgccgc agtggttcgt cagcatggat cagaaaggtc tgcgtgcgca gtcactgaaa
     3661 gagatcaaag gcgtgcagtg gatcccggac tggggccagg cgcgtatcga gtcgatggtt
     3721 gctaaccgtc ctgactggtg tatctcccgt cagcgcacct ggggtgtacc gatgtcactg
     3781 ttcgtgcaca aagacacgga agagctgcat ccgcgtaccc ttgaactgat ggaagaagtg
     3841 gcaaaacgcg ttgaagtcga tggcatccag gcgtggtggg atctcgatgc gaaagagatc
     3901 ctcggcgacg aagctgatca gtacgtgaaa gtgccggaca cattggatgt atggtttgac
     3961 tccggatcta cccactcttc tgttgttgac gtgcgtccgg aatttgccgg tcacgcagcg
     4021 gacatgtatc tggaaggttc tgaccaacac cgcggctggt tcatgtcttc cctaatgatc
     4081 tccaccgcga tgaagggtaa agcgccgtat cgtcaggtac tgacccacgg ctttaccgtg
     4141 gatggtcagg gccgcaagat gtctaaatcc atcggcaata ccgtttcgcc gcaggatgtg
     4201 atgaacaaac tgggcgcgga tattctgcgt ctgtgggtgg catcaaccga ctacaccggt
     4261 gaaatggccg tttctgacga gatcctgaaa cgtgctgccg atagctatcg tcgtatccgt
     4321 aacaccgcgc gcttcctgct ggcaaacctg aacggttttg atccagcaaa agatatggtg
     4381 aaaccggaag agatggtggt actggatcgc tgggccgtag gttgtgcgaa agcggcacag
     4441 gaagacatcc tcaaggcgta cgaagcatac gatttccacg aagtggtaca gcgtctgatg
     4501 cgcttctgct ccgttgagat gggttccttc tacctcgaca tcatcaaaga ccgtcagtac
     4561 accgccaaag cggacagtgt ggcgcgtcgt agctgccaga ctgcgctata tcacatcgca
     4621 gaagcgctgg tgcgctggat ggcaccaatc ctctccttca ccgctgatga agtgtggggc
     4681 tacctgccgg gcgaacgtga aaaatacgtc ttcaccggtg agtggtacga aggcctgttt
     4741 ggcctggcag acagtgaagc gatgaacgat gcgttctggg acgagctgtt gaaagtgcgt
     4801 ggcgaagtga acaaagtcat tgagcaagcg cgtgccgaca agaaagtggg tggctcgctg
     4861 gaagcggcag taaccttgta tgcagaaccg gaactgtcgg cgaaactgac cgcgctgggc
     4921 gatgaattac gatttgtcct gttgacctcc ggcgctaccg ttgcagacta taacgacgca
     4981 cctgctgatg ctcagcagag cgaagtactc aaagggctga aagtcgcgtt gagtaaagcc
     5041 gaaggtgaga agtgcccacg ctgctggcac tacacccagg atgtcggcaa ggtggcggaa
     5101 cacgcagaaa tctgcggccg ctgtgtcagc aacgtcgccg gtgacggtga aaaacgtaag
     5161 tttgcctgaa agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct
     5221 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     5281 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     5341 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5401 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5461 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5521 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5581 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5641 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5701 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5761 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     5821 cacttttagg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5881 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5941 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6001 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6061 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     6121 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6181 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6241 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6301 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6361 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6421 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6481 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6541 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6601 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6661 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6721 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6781 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6841 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6901 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6961 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     7021 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     7081 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     7141 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     7201 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     7261 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     7321 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     7381 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7441 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7501 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7561 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7621 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     7681 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     7741 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     7801 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     7861 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     7921 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     7981 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     8041 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     8101 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     8161 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttc
//
