LOCUS       sequence-240850-        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-TyrRS-His
FEATURES             Location/Qualifiers
     source          1..6635
                     /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"
     RBS             1443..1465
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1480..1504
                     /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(1504..1523)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1505..1523)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1607..1626)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1832..1909
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1910..2992
                     /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(1929..1948)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3801..3992
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3986..4008)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4094..4233
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4149..4168
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4248..4265)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4419..5007)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4499..4518)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5178..5969,5970..6038))
                     /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     5801..5820
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6039..6143)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6169..6624)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6306..6327)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6518..6537
                     /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 gagtttccag caaatcagac
      181 agtaattctt tttaccgcga cgcagtaagg taaaacgacc aaacagacga tcttcttctt
      241 taaagaagta ttcaggatcg gactgttttt caccgttaat ggtgatggca ttggaggcga
      301 tagttttacg tgcctgacca cgggaaggtt gcagttcaga atcgaccagt gcctgcatca
      361 ggtctgcgcc cttttccatc tcaaccatcg gtacgccgtc ctgcgccagc tgttcgaagt
      421 ccgcttcact cagcgcactc aaagaaccgc tgaacaggca ttcggtaata cgttttgccg
      481 cctgtaaacc ttcttcaccg tgaaccagac gagtcacctg ctccgccagt acatactggg
      541 cgcgcggtgc tttaccgctg tttttatctt cttcttccag ggcgttgatc tcttcaatgc
      601 tcataaaggt gaagaacttc aggaagcggt aaacgtcggc atccgcagtg ttgatccaga
      661 actggtagaa tttgtacggg ctggttttct tcggatccaa ccagactgcg ccgccttcag
      721 ttttaccaaa tttggtgcca tctgctttag tgatcagcgg aacggtcagg ccaaacacct
      781 gattctgatg cagacgacgg gtcaggtcga taccagaagt gatgttaccc cactggtcag
      841 aaccaccaat ttgcagcacc acaccgtact gtttgttcag acaggcgaag tcataaccct
      901 gcaacaggtt gtaggaaaac tcagtgaacg aaatcccctg atcttcacgg ttgagacgct
      961 gcttaaccgc ttctttgttg atcatctggt taacggagaa gtgtttgcca atatcgcgca
     1021 ggaaggtcag cacattcata ttgccgaacc agtcatagtt gttcgccgcg atagcagagt
     1081 tttctccaca gtcgaaatcg aggaacgggg caacctgctt acggattttg tccacccact
     1141 cctgaacagt ttcttcggtg ttcagcttac gctcggcagc tttgaagctc gggtcgccaa
     1201 tcagacccgt cgcgccgcct accagcgcaa ccggcttgtg gcccgcctgc tggaagcgtt
     1261 tcaggcataa caatggaaca agatgcccca aatgcaagct gtcagcggta ggatcgaagc
     1321 cgcaatagag cgcgatcggg ccttgcgcca gtcgctctgc taacgcttcc tcgtccgtca
     1381 cctgggctac cagcccccgc tcttgcaatt gtttaatcaa gttactgctt gccatatgta
     1441 tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa
     1501 ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct ctacgccgga
     1561 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     1621 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
     1681 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
     1741 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
     1801 gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg
     1861 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     1921 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     1981 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     2041 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     2101 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     2161 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     2221 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     2281 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     2341 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     2401 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     2461 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     2521 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     2581 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     2641 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     2701 tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     2761 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     2821 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     2881 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     2941 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     3001 caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag
     3061 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     3121 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
     3181 tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg
     3241 gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg
     3301 agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt
     3361 cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga
     3421 tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat
     3481 gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca
     3541 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     3601 ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
     3661 ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa
     3721 cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc
     3781 ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct
     3841 ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac
     3901 aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg
     3961 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     4021 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     4081 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     4141 taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg
     4201 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     4261 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4321 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4381 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4441 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4501 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4561 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4621 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4681 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4741 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4801 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     4861 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4921 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4981 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5041 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5101 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5161 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5221 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5281 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5341 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5401 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5461 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     5521 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5581 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5641 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5701 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5761 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5821 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5881 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5941 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6001 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6061 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6121 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     6181 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     6241 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     6301 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     6361 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     6421 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     6481 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     6541 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     6601 ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
