LOCUS       sequence-258637-        6365 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 6365)
  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 6365)
  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-TrpRS-His
FEATURES             Location/Qualifiers
     source          1..6365
                     /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)."
     RBS             1562..1584
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2600..2617
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2670..2688)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2684..2731
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2768..3223
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2855..2874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3065..3086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3249..3353
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3354..3422,3423..4214)
                     /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(3572..3591)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4385..4973
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4874..4893
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5127..5144
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5159..5298
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5224..5243)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5384..5406
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5400..5591)
                     /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 ctagaaataa
     1561 ttttgtttaa ctttaagaag gagatataca tatgactaag cccatcgttt ttagtggcgc
     1621 acagccctca ggtgaattga ccattggtaa ctacatgggt gcgctgcgtc agtgggtaaa
     1681 catgcaggat gactaccatt gcatttactg tatcgttgac caacacgcga tcaccgtgcg
     1741 ccaggatgca cagaagctgc gtaaagcgac gctggatacg ctggccttgt atctggcttg
     1801 tggtatcgat cctgagaaaa gcaccatttt tgttcagtcc cacgtgccgg aacatgcaca
     1861 gttaggctgg gcactgaact gctataccta cttcggcgaa ctgagtcgca tgacgcagtt
     1921 taaagataaa tctgcgcgtt atgccgagaa catcaacgct ggtctgtttg actatccggt
     1981 gctgatggca gcggacatcc tgctgtatca aactaatctg gtaccggtgg gtgaagacca
     2041 gaaacagcac ctcgaactga gccgcgatat tgcccagcgt ttcaacgcgc tgtatggcga
     2101 gatctttaag gtgccggagc cgtttattcc gaaatctggc gcgcgcgtaa tgtcgctgct
     2161 ggagccgacc aagaagatgt ccaagtctga cgataatcgc aataacgtta tcggcctgct
     2221 ggaagatccg aaatcggtag tgaagaaaat caaacgtgcg gtcactgact ccgacgagcc
     2281 gccggtagtt cgctacgatg tgcagaacaa agcgggcgtt tccaacctgt tggatatcct
     2341 ttcagcggta acgggccaga gcatcccaga actggaaaaa cagttcgaag gcaagatgta
     2401 tggtcatctg aaaggtgaag tggctgatgc cgtttccggt atgctgactg aattgcagga
     2461 acgctatcac cgtttccgca acgatgaagc cttcctgcaa caggtgatga aagatggcgc
     2521 ggaaaaagcc agcgcgcacg cttcccgtac gctaaaagcg gtgtacgaag cgattggttt
     2581 tgtggcgaag cgtctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
     2641 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     2701 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     2761 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2821 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2881 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2941 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3001 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3061 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3121 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3181 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     3241 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3301 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3361 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3421 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     3481 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3541 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     3601 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     3661 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3721 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3781 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3841 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     3901 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3961 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4021 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4081 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4141 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4201 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4261 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4321 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4381 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4441 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4501 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     4561 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4621 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4681 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4741 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4801 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4861 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4921 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4981 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5041 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5101 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5161 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     5221 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     5281 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5341 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5401 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     5461 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     5521 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     5581 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     5641 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     5701 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     5761 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     5821 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     5881 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     5941 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     6001 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     6061 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     6121 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     6181 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     6241 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     6301 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     6361 gccta
//
