LOCUS       sequence-258701-        6795 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 6795)
  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 6795)
  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: pET15b-His-RF2
FEATURES             Location/Qualifiers
     source          1..6795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1281..2363)
                     /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     2325..2344
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2364..2441)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2647..2666
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2750..2769
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2750..2768
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2769..2793
                     /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             2808..2830
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2849..2866
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2876..2893
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(4045..4063)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4059..4106
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4281..4286,4287..4303,4304..4309))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4336..4354)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4422..4526
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4527..4595,4596..5387)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4745..4764)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5558..6146
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6047..6066
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6300..6317
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6332..6471
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6397..6416)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6557..6579
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6573..6764)
                     /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 cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg
      481 gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc
      541 cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg
      601 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
      661 caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg
      721 cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga
      781 gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca
      841 acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc
      901 ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa
      961 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     1021 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     1081 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     1141 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     1201 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     1261 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1321 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     1381 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     1441 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     1501 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     1561 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     1621 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     1681 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     1741 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     1801 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     1861 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     1921 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     1981 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     2041 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     2101 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     2161 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     2221 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     2281 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     2341 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     2401 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     2461 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     2521 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     2581 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     2641 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     2701 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     2761 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     2821 aagaaggaga tataccatgg gcagcagcca tcatcatcat catcacagca gcggcctggt
     2881 gccgcgcggc agccatatgt ttgaaattaa tccggtaaat aatcgcattc aggacctcac
     2941 ggaacgctcc gacgttctta gggggtatct tgactacgac gccaagaaag agcgtctgga
     3001 agaagtaaac gccgagctgg aacagccgga tgtctggaac gaacccgaac gcgcacaggc
     3061 gctgggtaaa gagcgttcct ccctcgaagc cgttgtcgac accctcgacc aaatgaaaca
     3121 ggggctggaa gatgtttctg gtctgctgga actggctgta gaagctgacg acgaagaaac
     3181 ctttaacgaa gccgttgctg aactcgacgc cctggaagaa aaactggcgc agcttgagtt
     3241 ccgccgtatg ttctctggcg aatatgacag cgccgactgc tacctcgata ttcaggcggg
     3301 gtctggcggt acggaagcac aggactgggc gagcatgctt gagcgtatgt atctgcgctg
     3361 ggcagaatcg cgtggtttca aaactgaaat catcgaagag tcggaaggtg aagtggcggg
     3421 tattaaatcc gtgacgatca aaatctccgg cgattacgct tacggctggc tgcgtacaga
     3481 aaccggcgtt caccgcctgg tgcgtaaaag cccgtttgac tccggcggtc gtcgccacac
     3541 gtcgttcagc tccgcgtttg tttatccgga agttgatgat gatattgata tcgaaatcaa
     3601 cccggcggat ctgcgcattg acgtttatcg cacgtccggc gcgggcggtc agcacgttaa
     3661 ccgtaccgaa tctgcggtgc gtattaccca catcccgacc gggatcgtga cccagtgcca
     3721 gaacgaccgt tcccagcaca agaacaaaga tcaggccatg aagcagatga aagcgaagct
     3781 ttatgaactg gagatgcaga agaaaaatgc cgagaaacag gcgatggaag ataacaaatc
     3841 cgacatcggc tggggcagcc agattcgttc ttatgtcctt gatgactccc gcattaaaga
     3901 tctgcgcacc ggggtagaaa cccgcaacac gcaggccgtg ctggacggca gcctggatca
     3961 atttatcgaa gcaagtttga aagcagggtt atgaggatcc ggctgctaac aaagcccgaa
     4021 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     4081 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa
     4141 gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc
     4201 gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt
     4261 taactgtgat aaactaccgc attaaagctt atcgatgata agctgtcaaa catgagaatt
     4321 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     4381 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4441 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4501 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     4561 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     4621 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     4681 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     4741 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     4801 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     4861 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     4921 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     4981 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5041 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     5101 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5161 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5221 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     5281 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5341 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     5401 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5461 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5521 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5581 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5641 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5701 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5761 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5821 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5881 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5941 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6001 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6061 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6121 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6181 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6241 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     6301 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     6361 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     6421 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     6481 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6541 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     6601 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     6661 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     6721 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     6781 gggggatttc tgttc
//
