LOCUS       sequence-258617-        8351 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 8351)
  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 8351)
  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-GlyRS-His
FEATURES             Location/Qualifiers
     source          1..8351
                     /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             4586..4603
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4656..4674)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4670..4717
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4754..5209
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4841..4860)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5051..5072
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5235..5339
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5340..5408,5409..6200)
                     /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(5558..5577)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6371..6959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6860..6879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7113..7130
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7145..7284
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7210..7229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7370..7392
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7386..7577)
                     /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 tatgcaaaag tttgatacca ggaccttcca
     1621 gggcttgatc ctgaccttac aggattactg ggctcgccag ggctgcacca ttgttcaacc
     1681 attggacatg gaagtcggcg cgggaacctc tcacccaatg acctgtctgc gcgagctggg
     1741 gccagaaccg atggcggctg cttatgttca gccttctcgt cgcccgaccg atggtcgcta
     1801 cggcgaaaac cccaaccgtt tacagcacta ctatcagttc caggtggtca ttaagccatc
     1861 gccggacaat attcaggagc tgtacctcgg ttctctgaaa gagctgggca tggacccgac
     1921 tattcacgac atccgtttcg tggaagataa ctgggaaaac ccgacgctgg gtgcctgggg
     1981 actgggctgg gaagtgtggc tgaacggcat ggaagtgacg cagttcactt acttccagca
     2041 ggttggtggt ctggagtgta aaccggttac cggcgagatc acctacggtc tggaacgtct
     2101 ggccatgtac attcagggcg tagacagcgt ttacgacctg gtctggagcg acggcccgct
     2161 gggtaaaacc acctacggcg acgtgttcca tcagaacgaa gtggagcagt ccacttacaa
     2221 cttcgaatac gcggatgtgg acttcctgtt cacctgcttc gagcagtacg agaaagaagc
     2281 gcagcagctg ctggcgctgg aaaatccgct gccgctgcca gcctacgagc gtattctgaa
     2341 agccgcccac agcttcaacc tgctggatgc gcgtaaagcc atctccgtca ccgagcgtca
     2401 gcgctacatt ctgcgcattc gcaccctgac caaagcagtg gcagaagcat actacgcttc
     2461 ccgtgaagcc ctcggcttcc cgatgtgcaa caaagataag taagaggcgg ctatgtctga
     2521 gaaaactttt ctggtggaaa tcggcactga agagctgcca ccaaaagcac tgcgcagcct
     2581 ggctgagtcc tttgctgcga actttactgc ggagctggat aacgctggcc tcgcacacgg
     2641 caccgttcaa tggtttgctg ctccgcgtcg tctggcgctg aaagtagcta acctggcgga
     2701 agcgcaaccg gatcgtgaaa tcgaaaaacg cggcccggcg attgcccagg cgttcgacgc
     2761 tgaaggcaaa ccgagcaaag cggcagaagg ttgggcgcgt ggttgcggta ttaccgttga
     2821 ccaggctgag cgtctgacta ccgataaagg cgaatggctg ctgtatcgcg cccatgtgaa
     2881 gggcgaaagc accgaagcac tgctgccgaa tatggttgcg acttctctgg cgaaactgcc
     2941 gatcccgaaa ctgatgcgtt ggggcgcaag cgacgtgcac ttcgtgcgtc cggtgcacac
     3001 cgtgaccctg ctgctgggcg acaaagtcat tccggcaacc attctgggca ttcagtccga
     3061 tcgcgtgatt cgcggccacc gctttatggg cgagccggaa ttcaccatcg ataacgccga
     3121 tcagtatccg gaaattctgc gtgagcgtgg gaaagtcatc gccgattacg aagaacgtaa
     3181 ggcgaagatt aaagccgatg ccgaagaagc agcgcgtaag attggcggta acgctgactt
     3241 aagcgaaagc ctgctggaag aagtggcttc gctggtggag tggccggtcg ttctgaccgc
     3301 aaaattcgaa gagaaattcc tcgcggtgcc ggctgaagcg ctggtttaca ccatgaaagg
     3361 tgaccagaaa tacttcccgg tgtatgcgaa cgacggcaaa ctgctgccga actttatctt
     3421 cgttgccaac atcgaatcga aagatccgca gcagattatc tccggtaacg agaaagtcgt
     3481 tcgtccgcgt ctggcggatg ccgagttctt cttcaacacc gaccgtaaaa aacgtcttga
     3541 agataacctg ccgcgcctgc aaaccgtgtt gttccagcaa cagttgggga cgctgcgcga
     3601 caaaactgac cgcatccagg cgctggctgg ctggattgct gaacagattg gcgctgacgt
     3661 taaccacgct acccgtgcgg gtctgctgtc taagtgcgac ctgatgacca acatggtctt
     3721 cgagttcacc gacacccagg gcgttatggg gatgcactat gcgcgtcacg atggcgaagc
     3781 ggaagatgtc gcggtggcgc tgaatgagca gtatcagccg cgttttgctg gtgatgacct
     3841 gccgtccaac ccagtagctt gtgcgctggc gattgctgac aagatggata ccctggcggg
     3901 tatcttcggt atcggtcagc atccgaaagg cgacaaagac ccgtttgcgc tgcgtcgtgc
     3961 cgcgcttggc gtgctgcgaa ttatcgttga gaagaacctc aaccttgatc tgcaaacgct
     4021 gaccgaagaa gcggtgcgtc tgtatggcga taagctgact aatgccaacg tagttgatga
     4081 tgttatcgac tttatgctcg gtcgcttccg cgcctggtat caggacgaag gttataccgt
     4141 tgacaccatc caggcggtac tggcgcgtcg tccgactcgt ccggctgatt tcgatgcccg
     4201 tatgaaagcg gtatcgcatt tccgtaccct ggatgcagct gctgcactgg cggcggcgaa
     4261 caaacgtgta tctaacattc tggcgaaatc tgacgaagtg ctgagcgacc gcgtgaatgc
     4321 ctctaccctg aaagagccgg aagaaattaa actggcgatg caggttgtgg tgctacgtga
     4381 caagctggag ccgtacttta cggaaggtcg ttaccaggat gcgctggtcg aactggctga
     4441 gctgcgtgaa ccggttgatg ctttcttcga taaagtgatg gtcatggttg atgacaaaga
     4501 attgcgtatc aaccgtctga ccatgctgga gaaactgcgc gaactgttcc tgcgcgttgc
     4561 ggatatttcg ctgttgcaac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     4621 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     4681 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     4741 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4801 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4861 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4921 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4981 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5041 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5101 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5161 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5221 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5281 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5341 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5401 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5461 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5521 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5581 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5641 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5701 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5761 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5821 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5881 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5941 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6001 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6061 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6121 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6181 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6241 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6301 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6361 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6421 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6481 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     6541 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6601 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6661 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6721 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6781 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6841 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6901 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6961 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     7021 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     7081 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     7141 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     7201 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     7261 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     7321 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     7381 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     7441 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     7501 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     7561 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     7621 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     7681 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     7741 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     7801 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     7861 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     7921 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     7981 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     8041 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     8101 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     8161 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     8221 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     8281 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     8341 cccggtgcct a
//
