LOCUS       sequence-258624-        7920 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 7920)
  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 7920)
  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-LeuRS-His
FEATURES             Location/Qualifiers
     source          1..7920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(35..1117)
                     /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     1079..1098
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1118..1195)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1401..1420
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1504..1523
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1504..1522
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1523..1547
                     /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)."
     CDS             4155..4172
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4225..4243)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4239..4286
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4323..4778
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4410..4429)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4620..4641
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4804..4908
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4909..4977,4978..5769)
                     /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(5127..5146)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5940..6528
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6429..6448
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6682..6699
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6714..6853
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6779..6798)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6939..6961
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6955..7146)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
       61 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
      121 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
      181 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
      241 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
      301 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
      361 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
      421 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
      481 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
      541 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
      601 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
      661 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
      721 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
      781 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
      841 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
      901 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
      961 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     1021 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     1081 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     1141 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     1201 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     1261 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     1321 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     1381 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     1441 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     1501 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaggag
     1561 atgctgccat gcaagagcaa taccgcccgg aagagataga atccaaagta cagcttcatt
     1621 gggatgagaa gcgcacattt gaagtaaccg aagacgagag caaagagaag tattactgcc
     1681 tgtctatgct tccctatcct tctggtcgac tacacatggg ccacgtacgt aactacacca
     1741 tcggtgacgt gatcgcccgc taccagcgta tgctgggcaa aaacgtcctg cagccgatcg
     1801 gctgggacgc gtttggtctg cctgcggaag gcgcggcggt gaaaaacaac accgctccgg
     1861 caccgtggac gtacgacaac atcgcgtata tgaaaaacca gctcaaaatg ctgggctttg
     1921 gttatgactg gagccgcgag ctggcaacct gtacgccgga atactaccgt tgggaacaga
     1981 aattcttcac cgagctgtat aaaaaaggcc tggtatataa gaagacttct gcggtcaact
     2041 ggtgcccgaa cgaccagacc gtactggcga acgaacaagt tatcgacggc tgctgctggc
     2101 gctgcgatac caaagttgaa cgtaaagaga tcccgcagtg gtttatcaaa atcactgctt
     2161 acgctgacga gctgctcaac gatctggata aactggatca ctggccagac accgttaaaa
     2221 ccatgcagcg taactggatc ggtcgttccg aaggcgtgga gatcaccttc aacgttaacg
     2281 actatgacaa cacgctgacc gtttacacta cccgcccgga cacctttatg ggttgtacct
     2341 acctggcggt agctgcgggt catccgctgg cgcagaaagc ggcggaaaat aatcctgaac
     2401 tggcggcctt tattgacgaa tgccgtaaca ccaaagttgc cgaagctgaa atggcgacga
     2461 tggagaaaaa aggcgtcgat actggcttta aagcggttca cccattaacg ggcgaagaaa
     2521 ttcccgtttg ggcagcaaac ttcgtattga tggagtacgg cacgggcgca gttatggcgg
     2581 taccggggca cgaccagcgc gactacgagt ttgcctctaa atacggcctg aacatcaaac
     2641 cggttatcct ggcagctgac ggctctgagc cagatctttc tcagcaagcc ctgactgaaa
     2701 aaggcgtgct gttcaactct ggcgagttca acggtcttga ccatgaagcg gccttcaacg
     2761 ccatcgccga taaactgact gcgatgggcg ttggcgagcg taaagtgaac taccgcctgc
     2821 gcgactgggg tgtttcccgt cagcgttact ggggcgcgcc gattccgatg gtgacgctgg
     2881 aagacggtac cgtaatgccg accccggacg accagctgcc ggtgatcctg ccggaagatg
     2941 tggtaatgga cggcattacc agcccgatta aagcagatcc ggagtgggcg aaaactaccg
     3001 ttaacggtat gccagcactg cgtgaaaccg acactttcga cacctttatg gagtcctcct
     3061 ggtactatgc gcgctacact tgcccgcagt acaaagaagg tatgctggat tccgaagcgg
     3121 ctaactactg gctgccggtg gatatctaca ttggtggtat tgaacacgcc attatgcacc
     3181 tgctctactt ccgcttcttc cacaaactga tgcgtgatgc aggcatggtg aactctgacg
     3241 aaccagcgaa acagttgctg tgtcagggta tggtgctggc agatgccttc tactatgttg
     3301 gcgaaaacgg cgaacgtaac tgggtttccc cggttgatgc tatcgttgaa cgtgacgaga
     3361 aaggccgtat cgtgaaagcg aaagatgcgg caggccatga actggtttat accggcatga
     3421 gcaaaatgtc caagtcgaag aacaacggta tcgacccgca ggtgatggtt gaacgttacg
     3481 gcgcggacac cgttcgtctg tttatgatgt ttgcttctcc ggctgatatg actctcgaat
     3541 ggcaggaatc cggtgtggaa ggggctaacc gcttcctgaa acgtgtctgg aaactggttt
     3601 acgagcacac agcaaaaggt gatgttgcgg cactgaacgt tgatgcgctg actgaaaatc
     3661 agaaagcgct gcgtcgcgat gtgcataaaa cgatcgctaa agtgaccgat gatatcggcc
     3721 gtcgtcagac cttcaacacc gcaattgcgg cgattatgga gctgatgaac aaactggcga
     3781 aagcaccaac cgatggcgag caggatcgcg ctctgatgca ggaagcactg ctggccgttg
     3841 tccgtatgct taacccgttc accccgcaca tctgcttcac gctgtggcag gaactgaaag
     3901 gcgaaggcga tatcgacaac gcgccgtggc cggttgctga cgaaaaagcg atggtggaag
     3961 actccacgct ggtcgtggtg caggttaacg gtaaagtccg tgccaaaatc accgttccgg
     4021 tggacgcaac ggaagaacag gttcgcgaac gtgctggcca ggaacatctg gtagcaaaat
     4081 atcttgatgg cgttactgta cgtaaagtga tttacgtacc aggtaaactc ctcaatctgg
     4141 tcgttggcct cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     4201 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     4261 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     4321 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4381 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4441 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4501 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     4561 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4621 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4681 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4741 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     4801 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4861 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4921 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4981 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5041 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5101 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5161 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5221 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5281 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5341 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5401 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg
     5461 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5521 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5581 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5641 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5701 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5761 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5821 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5881 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5941 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6001 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6061 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     6121 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6181 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6241 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6301 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6361 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6421 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6481 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6541 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6601 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6661 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     6721 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     6781 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     6841 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     6901 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     6961 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     7021 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     7081 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     7141 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     7201 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     7261 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     7321 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     7381 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     7441 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     7501 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     7561 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     7621 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     7681 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     7741 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     7801 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     7861 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
//
