LOCUS       sequence-240858-        7371 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 7371)
  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 7371)
  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-MetRS-His
FEATURES             Location/Qualifiers
     source          1..7371
                     /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    2216..2240
                     /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(2240..2259)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2241..2259)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2343..2362)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2568..2645
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2646..3728
                     /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(2665..2684)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4537..4728
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4722..4744)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4830..4969
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4885..4904
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4984..5001)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5155..5743)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5235..5254)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5914..6705,6706..6774))
                     /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     6537..6556
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6775..6879)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6905..7360)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7042..7063)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7254..7273
                     /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 gagtttcacc tgatgacccg
      181 gtttagcacc ggcatccggg cttagcagga aaatatcttt cccgccagga ccggcagcca
      241 tcaccatgcc ttcagagata ccgaagcgca ttttacgtgg tgccaggtta gccaccataa
      301 tggtgtgacg accaatcagt gcctgcggat ccgggtaagc agaacgaata ccggagaaga
      361 cattgcgttt ttcaccgccg agatccagcg tcaggcgcag cagtttgtca gaaccttcaa
      421 caaactctgc gttttcaatc agcgccacgc gcaggtcaac tttagcgaag tcgtcaaagg
      481 tgatggtttc ctgaatcgga tcatctgcca gcgggccagt taccggcgcg gcagcggctt
      541 ttacttcttc tttagaggct tccaccagtg cttcaacctg cctcatatcg atgcggttat
      601 acagcgcctt gaacggattc actttgtggc ccagcagcgg ttgctggata ccatcccagg
      661 tcagttccgt attgaggaat gcttctgcac gctcggtcag tttcggcagt accggcttca
      721 ggtaagtcat cagcacgcgg aacaggttga tgcccattga gcaaattgcc tgcaggtcgg
      781 catcgcggcc ttcctgtttc gccaccaccc acggagcctg ttcatcgaca tagcggttag
      841 ccagatcagc cagcgccatg atttcgcgca cggctttacc aaattcacgg ctttcccacg
      901 cttcaccaat cacttcagcg gcatcagtga aggttttgta caactgcggg tcagccagtt
      961 cgcttgccag cacgccgtca aaacgcttgt tgataaagcc cgcattacgg gaggccaggt
     1021 taaccacttt gttaacgata tcggcattca cacgctgaac gaaatcttcc aggttgagat
     1081 cgatatcatc aatgcgcgaa gagagtttcg cagtgtagta gtaacgcagg ctgtctgcgt
     1141 caaaatgatt cagccaggtg ctggctttaa taaaggtgcc gcgagacttg gacatctttg
     1201 cgccgttcac cgtcacatag ccatgaacaa acaggttgga cggcttgcgg aagttgctgc
     1261 cttccagcat ggcaggccag aacaggctgt ggaagtaaac aatatcttta ccgatgaagt
     1321 ggtacagctc ggcggtggag tctttcttcc agtattcatc gaagcttacg ctgtcgccgc
     1381 gcttgtcgca cagattcttg aaagaaccca tgtagccaat cggtgcgtcc agccagacgt
     1441 agaaatattt gcccggcgcg ttcggaattt caaaaccgaa gtaaggggcg tcgcgggaga
     1501 tatcccactg ttgcaggcca gattcaaacc actcctgcat tttatttgcc acctgctcct
     1561 gcaacgcacc gctgcgggtc catgcctgca acatttcgct gaaagagggc agatcaaaga
     1621 agaagtgttc agaatcacgc attaccggcg tagcgccaga aaccaccgat ttcggctcga
     1681 tcagttcagt cgggctgtag gtcgcgccgc agacttcgca gttatcgccg tattgatccg
     1741 gggatttaca tttcgggcag gtgcctttca caaaacggtc cggcaggaac atgccttttt
     1801 ccggatcgta cagctgagag atggtgcggt ttttaataaa accgttttct ttcaggcgag
     1861 agtagataag ttctgacaac tggcggttct cttcgctgtg cgtcgagtga tagttgtcat
     1921 agctgatgtt aaagcctgcg aaatcagtct gatgctcctg actcatttcg ccaatcatct
     1981 gctccggggt gataccaagc tgctgagctt tcagcatgat cggtgtaccg tgggcatcgt
     2041 cggcgcagat gaagttgacc tcgtggccgc gcattcgctg gtaacggacc cagacatcag
     2101 cctggatgtg ctccagcata tggccgaggt ggattgagcc gttagcgtac ggcagtgcgc
     2161 acgtcaccag aattttcttc gcgacttgag tcatagtagg atctccttct agaggggaat
     2221 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc
     2281 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     2341 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     2401 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     2461 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     2521 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
     2581 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     2641 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     2701 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     2761 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     2821 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     2881 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     2941 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     3001 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     3061 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     3121 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     3181 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     3241 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     3301 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     3361 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     3421 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     3481 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     3541 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     3601 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     3661 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     3721 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
     3781 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     3841 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     3901 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     3961 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     4021 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc
     4081 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     4141 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     4201 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     4261 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
     4321 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
     4381 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
     4441 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
     4501 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
     4561 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
     4621 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
     4681 taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     4741 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     4801 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     4861 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt
     4921 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     4981 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5041 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5101 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5161 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5221 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5281 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5341 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5401 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5461 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5521 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5581 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5641 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5701 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5761 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5821 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5881 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5941 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6001 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6061 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6121 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6181 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt
     6241 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6301 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6361 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6421 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6481 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6541 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6601 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6661 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6721 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6781 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6841 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6901 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     6961 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     7021 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     7081 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     7141 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     7201 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     7261 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     7321 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc a
//
