LOCUS       sequence-258516-        6746 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 6746)
  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 6746)
  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-CysRS-His
FEATURES             Location/Qualifiers
     source          1..6746
                     /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             3724..3741
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3794..3812)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3808..3855
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3892..4347
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3979..3998)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4189..4210
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4373..4477
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4478..4546,4547..5338)
                     /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(4696..4715)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5509..6097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5998..6017
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6251..6268
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6283..6422
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6348..6367)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6508..6530
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6524..6715)
                     /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 acatatgcta
     2341 aaaatcttca atactctgac acgccaaaaa gaggaattta agcctattca cgccggggaa
     2401 gtcggcatgt acgtgtgtgg aatcaccgtt tacgatctct gtcatatcgg tcacgggcgt
     2461 acctttgttg cttttgacgt ggttgcgcgc tatctgcgtt tcctcggcta taaactgaag
     2521 tatgtgcgca acattaccga tatcgacgac aaaatcatca aacgcgccaa tgaaaatggc
     2581 gaaagctttg tggcgatggt ggatcgcatg atcgccgaaa tgcacaaaga ttttgatgct
     2641 ttgaacattc tgcgcccgga tatggagccg cgcgcgacgc accatatcgc agaaattatt
     2701 gaactcactg aacaactgat cgccaaaggt cacgcttatg tggcggacaa cggcgacgtg
     2761 atgttcgacg tcccgaccga tccaacttat ggcgtgctgt cgcgtcagga tctcgaccag
     2821 ctgcaggcag gcgcgcgcgt tgacgtggtc gacgacaaac gcaacccaat ggacttcgtt
     2881 ctgtggaaga tgtcgaaaga gggcgaaccg agctggccgt ctccgtgggg cgcgggtcgt
     2941 cctggctggc acattgaatg ttcggcaatg aactgcaagc agctgggtaa ccactttgat
     3001 atccacggcg gcggttcaga cctgatgttc ccgcaccacg aaaacgaaat cgcgcagtcc
     3061 acctgtgccc atgatggtca gtatgtgaac tactggatgc actcggggat ggtgatggtt
     3121 gaccgcgaga agatgtccaa atcgctgggt aacttcttta ccgtgcgcga tgtgctgaaa
     3181 tactacgacg cggaaaccgt gcgttacttc ctgatgtcgg gccactatcg cagccagttg
     3241 aactacagcg aagagaacct gaagcaggcg cgtgcggcgc tggagcgtct ctacactgcg
     3301 ctgcgcggca cagataaaac cgttgcgcct gccggtggcg aagcgtttga agcgcgcttt
     3361 attgaagcga tggacgacga tttcaacacc ccggaagcct attccgtact gtttgatatg
     3421 gcgcgtgaag taaaccgtct gaaagcagaa gatatggcag cggcgaatgc aatggcatct
     3481 cacctgcgta aactttccgc tgtattgggc ctgctggagc aagaaccgga agcgttcctg
     3541 caaagcggcg cgcaggcaga cgacagcgaa gtggctgaga ttgaagcgtt aattcaacag
     3601 cgtctggatg cccgtaaagc gaaagactgg gcggcggcgg atgcggcgcg tgatcgtctt
     3661 aacgagatgg ggatcgtgct ggaagatggc ccgcaaggga ccacctggcg tcgtaagctc
     3721 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     3781 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     3841 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     3901 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3961 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4021 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4081 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     4141 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4201 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     4261 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4321 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     4381 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4441 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4501 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4561 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4621 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4681 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4741 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4801 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4861 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4921 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4981 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     5041 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5101 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5161 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5221 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5281 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5341 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5401 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5461 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5521 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5581 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5641 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5701 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5761 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5821 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5881 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5941 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6001 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6061 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6121 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     6181 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     6241 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     6301 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     6361 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     6421 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     6481 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     6541 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     6601 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     6661 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     6721 cctccgtgta agggggattt ctgttc
//
