LOCUS       sequence-258599-        7133 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 7133)
  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 7133)
  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-AspRS-His
FEATURES             Location/Qualifiers
     source          1..7133
                     /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             4111..4128
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4181..4199)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4195..4242
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4279..4734
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4366..4385)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4576..4597
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4760..4864
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4865..4933,4934..5725)
                     /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(5083..5102)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5896..6484
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6385..6404
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6638..6655
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6670..6809
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6735..6754)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6895..6917
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6911..7102)
                     /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 acatatgcgt
     2341 acagaatatt gtggacagct ccgtttgtcc cacgtggggc agcaggtgac tctgtgtggt
     2401 tgggtcaacc gtcgtcgtga tcttggtagc ctgatcttca tcgatatgcg cgaccgcgaa
     2461 ggtatcgtgc aggtattttt cgatccggat cgtgcggacg cgttaaagct ggcctctgaa
     2521 ctgcgtaatg agttctgcat tcaggtcacg ggcaccgtac gtgcgcgtga cgaaaaaaat
     2581 attaaccgcg atatggcgac cggcgaaatc gaagtgctgg cgtcctcgct gactatcatc
     2641 aaccgcgcag atgttctgcc gcttgactct aaccacgtca acaccgaaga agcgcgtctg
     2701 aaataccgct acctcgacct gcgtcgtccg gaaatggctc agcgcctgaa aacccgcgct
     2761 aaaatcacca gcctggtgcg ccgttttatg gatgaccacg gcttcctcga catcgaaact
     2821 ccgatgctga ccaaagccac gccggaaggc gcgcgtgact acctggtgcc ttctcgtgtg
     2881 cacaaaggta aattctacgc actgccgcaa tccccgcagt tgttcaaaca gctgctgatg
     2941 atgtccggtt ttgaccgtta ctatcagatc gttaaatgct tccgtgacga agacctgcgt
     3001 gctgaccgtc agcctgaatt tactcagatc gatgtggaaa cttctttcat gaccgcgccg
     3061 caagtgcgtg aagtgatgga agcgctggtg cgtcatctgt ggctggaagt gaagggtgtg
     3121 gatctgggcg atttcccggt aatgaccttt gcggaagcag aacgccgtta tggttctgat
     3181 aaaccggatc tgcgtaaccc gatggaactg actgacgttg ctgatctgct gaaatctgtt
     3241 gagtttgctg tatttgcagg tccggcgaac gatccgaaag gtcgcgtagc ggctctgcgc
     3301 gttccgggcg gcgcatcgct gacccgtaag cagatcgacg aatacggtaa cttcgttaaa
     3361 atctacggcg cgaaaggtct ggcttacatc aaagttaacg aacgcgcgaa aggtctggaa
     3421 ggtatcaaca gcccggtagc gaagttcctt aatgcagaaa tcatcgaaga catcctggat
     3481 cgtactgccg cgcaagatgg cgatatgatt ttcttcggtg ccgacaacaa gaaaattgtt
     3541 gccgacgcga tgggtgcact gcgcctgaaa gtgggtaaag accttggtct gaccgacgaa
     3601 agcaaatggg caccgctgtg ggttatcgac ttcccgatgt ttgaagacga cggtgaaggc
     3661 ggcctgacgg caatgcacca tccgttcacc tcaccgaaag atatgacggc tgcagaactg
     3721 aaagctgcac cggaaaatgc ggtggcgaac gcttacgata tggtcatcaa tggttacgaa
     3781 gtgggcggtg gttcagtacg tatccataat ggtgatatgc agcagacggt gtttggtatt
     3841 ctgggtatca acgaagagga acagcgcgag aaattcggct tcctgctcga cgctctgaaa
     3901 tacggtactc cgccgcacgc aggtctggca ttcggtcttg accgtctgac catgctgctg
     3961 accggcaccg acaatatccg tgacgttatc gccttcccga aaaccacggc ggcagcgtgt
     4021 ctgatgactg aagcaccgag ctttgctaac ccgactgcac tggctgagct gagcattcag
     4081 gttgtgaaga aggctgagaa taacctcgag caccaccacc accaccactg agatccggct
     4141 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     4201 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     4261 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4321 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4381 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4441 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     4501 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4561 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     4621 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4681 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     4741 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4801 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4861 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4921 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4981 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5041 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5101 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5161 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5221 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5281 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5341 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5401 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5461 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5521 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5581 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     5641 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     5701 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     5761 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5821 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5881 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5941 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6001 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     6061 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6121 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6181 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6241 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6301 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6361 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6421 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6481 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6541 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     6601 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     6661 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     6721 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     6781 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     6841 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     6901 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     6961 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     7021 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     7081 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttc
//
