LOCUS       sequence-258619-        6635 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 6635)
  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 6635)
  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-HisRS-His
FEATURES             Location/Qualifiers
     source          1..6635
                     /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             2870..2887
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2940..2958)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2954..3001
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3038..3493
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3125..3144)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3335..3356
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3519..3623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3624..3692,3693..4484)
                     /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(3842..3861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4655..5243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5144..5163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5397..5414
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5429..5568
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5494..5513)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5654..5676
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5670..5861)
                     /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 tatggcaaaa aacattcaag ccattcgcgg
     1621 catgaacgat tacctgcctg gcgaaacggc catctggcag cgcattgaag gcacactgaa
     1681 aaacgtgctc ggcagctacg gttacagtga aatccgcttg ccgattgtag agcagacccc
     1741 gctattcaaa cgtgcgattg gtgaagtcac cgacgtggtt gaaaaagaga tgtacacctt
     1801 tgaggatcgc aatggcgaca gcctgactct gcgccctgaa gggacggcgg gctgtgtacg
     1861 cgccggcatc gagcatggtc ttctgtacaa tcaggaacag cgtctgtggt atatcgggcc
     1921 gatgttccgt cacgagcgtc cgcagaaagg gcgttatcgt cagttccatc agttgggctg
     1981 cgaagttttc ggtctgcaag gtccggatat cgacgctgaa ctgattatgc tcactgcccg
     2041 ctggtggcgc gcgctgggta tttccgagca cgtaactctt gagctgaact ctatcggttc
     2101 gctggaagca cgcgccaatt accgcgatgc gctggtggca ttccttgagc agcataaaga
     2161 aaagctggac gaagactgca aacgccgcat gtacactaac ccgctgcgcg tgctggattc
     2221 aaaaaatccg gaagtgcagg cgcttctcaa cgacgctccg gcattaggtg actatctgga
     2281 cgaggaatct cgtgagcatt ttgccggtct gtgcaaactg ctggagagcg cggggatcgc
     2341 ttacaccgta aaccagcgtc tggtgcgtgg tctggattac tacaaccgta ccgttttcga
     2401 gtgggtgact aacagtctcg gctcccaggg caccgtgtgt gcaggcggtc gttatgacgg
     2461 tcttgtggaa caactgggcg gtcgtgcaac accggctgtc ggttttgcta tgggcctcga
     2521 acgtcttgta ttgttagtac aggccgttaa tccggaattt aaagccgatc ctgttgtcga
     2581 tatatacctg gtggcttcag gtgctgatac acaatctgcg gctatggcat tagctgagcg
     2641 tctgcgtgat gaattaccgg gcgtgaaatt gatgaccaac cacggcggcg gcaactttaa
     2701 gaaacagttt gcccgtgctg ataaatgggg tgcccgcgtt gctgtggtgc tgggtgagtc
     2761 tgaagtggct aacggcacag cagtagtgaa ggatttgcgc tctggtgagc aaacggcagt
     2821 tgcgcaggat agcgtagccg cgcatttgcg cacgttactg ggtctcgagc accaccacca
     2881 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     2941 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     3001 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     3061 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3121 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3181 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3241 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3301 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3361 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3421 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     3481 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3541 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3601 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3661 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3721 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3781 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3841 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3901 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3961 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4021 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4081 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4141 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     4201 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4261 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4321 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4381 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4441 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4501 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4561 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4621 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4681 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4741 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4801 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4861 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4921 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4981 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5041 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5101 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5161 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5221 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5281 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5341 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5401 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     5461 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5521 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     5581 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5641 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5701 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     5761 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     5821 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     5881 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5941 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     6001 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     6061 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     6121 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     6181 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     6241 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     6301 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     6361 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     6421 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     6481 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     6541 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     6601 gctctcaagg gcatcggtcg agatcccggt gccta
//
