LOCUS       sequence-258308-        7025 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 7025)
  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 7025)
  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-GlnRS-His
FEATURES             Location/Qualifiers
     source          1..7025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..140
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(66..85)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     226..248
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(242..433)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1242..2324)
                     /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     2286..2305
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2325..2402)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2608..2627
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2711..2730
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2711..2729
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2730..2754
                     /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             2769..2791
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4467..4484
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4537..4555)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4551..4598
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4635..5090
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4722..4741)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4932..4953
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5116..5220
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5221..5289,5290..6081)
                     /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(5439..5458)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6252..6840
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6741..6760
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6994..7011
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
       61 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
      121 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
      181 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
      241 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
      301 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
      361 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
      421 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
      481 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
      541 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
      601 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
      661 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
      721 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
      781 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
      841 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
      901 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
      961 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     1021 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     1081 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     1141 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     1201 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     1261 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1321 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     1381 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     1441 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     1501 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     1561 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     1621 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     1681 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     1741 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     1801 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     1861 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     1921 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     1981 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     2041 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     2101 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     2161 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     2221 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     2281 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     2341 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     2401 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     2461 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     2521 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     2581 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     2641 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     2701 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     2761 gaaataattt tgtttaactt taagaaggag atatacatat gagtgaggca gaagcccgcc
     2821 cgactaactt tatccgtcag atcatcgatg aagatctggc cagtggtaag cacaccacag
     2881 tacacacccg tttcccgccg gagccgaatg gctatctgca tattggccat gcgaaatcta
     2941 tctgcctgaa cttcgggatc gcccaggact ataaaggcca gtgcaacctg cgtttcgacg
     3001 acactaaccc ggtaaaagaa gatatcgagt atgttgagtc gatcaaaaac gacgtagagt
     3061 ggttaggttt tcactggtct ggtaacgtcc gttactcctc cgattatttt gatcagctcc
     3121 acgcctatgc gatcgaactg atcaataaag gcctggcgta cgttgatgaa ctgacgccgg
     3181 aacagatccg cgaataccgc ggcaccctga cgcaaccggg taaaaacagc ccgtaccgcg
     3241 accgcagcgt tgaagagaac ctggcgctgt tcgaaaaaat gcgtgccggt ggttttgaag
     3301 aaggtaaagc ctgcctgcgt gcgaaaatcg acatggcttc accgtttatc gtgatgcgcg
     3361 atccggtgct gtaccgtatt aaatttgctg aacaccacca gactggcaac aagtggtgca
     3421 tctacccgat gtacgacttc acccactgca tcagcgatgc cctggaaggt attacgcact
     3481 ctctgtgtac gcttgagttc caggacaacc gtcgtctgta cgactgggta ctggacaaca
     3541 tcacgattcc tgttcacccg cgccagtatg agttctcgcg cctgaatctg gaatacaccg
     3601 tgatgtccaa gcgtaagttg aacctgctgg tgaccgacaa gcacgttgaa ggctgggatg
     3661 acccgcgtat gccgaccatt tccggtctgc gtcgtcgtgg ttacactgcg gcttctattc
     3721 gtgagttctg caaacgcatc ggcgtgacca agcaggacaa caccattgag atggcgtcgc
     3781 tggaatcctg catccgtgaa gatctcaacg aaaatgcgcc gcgcgcaatg gcggttatcg
     3841 atccggtgaa actggttatc gaaaactatc agggcgaagg cgaaatggtt accatgccga
     3901 accatccgaa caaaccggaa atgggcagcc gtcaggtgcc gtttagcggt gagatttgga
     3961 ttgatcgcgc cgatttccgc gaagaagcta acaagcagta caaacgtctg gtgctgggta
     4021 aagaagtgcg tctgcgtaat gcttatgtga ttaaggcaga acgcgtcgag aaagatgccg
     4081 aaggtaatat caccaccatc ttctgtactt atgacgccga taccttaagc aaagatccgg
     4141 cagatggtcg taaagtcaaa ggtgttattc actgggtgag cgcggcacat gcgctgccgg
     4201 ttgaaatccg tttgtatgac cgtctgttca gcgtgcctaa cccaggtgct gcggatgatt
     4261 tcctgtcggt gattaacccg gaatcgctgg tgatcaaaca gggctttgct gaaccgtcgc
     4321 tgaaagatgc ggttgcgggt aaagcattcc agtttgagcg tgaaggttac ttctgcctcg
     4381 atagccgcca ttctacggcg gaaaaaccgg tatttaaccg caccgttggg ctgcgtgata
     4441 cctgggcgaa agtaggcgag ctcgagcacc accaccacca ccactgagat ccggctgcta
     4501 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     4561 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     4621 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4681 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4741 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4801 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     4861 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4921 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     4981 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5041 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     5101 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5161 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5221 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5281 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5341 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5401 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5461 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5521 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5581 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5641 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5701 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5761 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5821 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5881 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5941 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6001 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6061 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6121 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6181 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6241 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6301 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6361 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     6421 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6481 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6541 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6601 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6661 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6721 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6781 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6841 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6901 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     6961 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     7021 gagcg
//
