LOCUS       sequence-258635-        6630 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 6630)
  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 6630)
  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-SerRS-His
FEATURES             Location/Qualifiers
     source          1..6630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(778..1860)
                     /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     1822..1841
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1861..1938)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2144..2163
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2247..2266
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2247..2265
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2266..2290
                     /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)."
     CDS             3608..3625
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3678..3696)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3692..3739
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3776..4231
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3863..3882)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4073..4094
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4257..4361
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4362..4430,4431..5222)
                     /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(4580..4599)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5393..5981
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5882..5901
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6135..6152
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6167..6306
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6232..6251)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6392..6414
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6408..6599)
                     /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 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
      481 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
      541 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
      601 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
      661 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
      721 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
      781 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      841 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
      901 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
      961 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1021 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     1081 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1141 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1201 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     1261 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     1321 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     1381 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     1441 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     1501 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     1561 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1621 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1681 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1741 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     1801 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     1861 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1921 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1981 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     2041 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     2101 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     2161 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     2221 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     2281 taacaattcc cctctagaag gagatataag catgctcgat cccaatctgc tgcgtaatga
     2341 gccagacgca gtcgctgaaa aactggcacg ccggggcttt aagctggatg tagataagct
     2401 gggcgctctt gaagagcgtc gtaaagtatt gcaggtcaaa acggaaaacc tgcaagcgga
     2461 gcgtaactcc cgatcgaaat ccattggcca ggcgaaagcg cgcggggaag atatcgagcc
     2521 tttacgtctg gaagtgaaca aactgggcga agagctggat gcagcaaaag ccgagctgga
     2581 tgctttacag gctgaaattc gcgatatcgc gctgaccatc cctaacctgc ctgcagatga
     2641 agtgccggta ggtaaagacg aaaatgacaa cgttgaagtc agccgctggg gtaccccgcg
     2701 tgagtttgac tttgaagttc gtgaccacgt gacgctgggt gaaatgcact ctggcctcga
     2761 ctttgcagct gcagttaagc tgactggttc ccgctttgtg gtaatgaaag ggcagattgc
     2821 tcgcatgcac cgcgcactgt cgcagtttat gctggatctg cataccgaac agcatggcta
     2881 cagtgagaac tatgttccgt acctggttaa ccaggacacg ctgtacggta cgggtcaact
     2941 gccgaaattt gctggcgatc tgttccatac tcgtccgctg gaagaagaag cagacaccag
     3001 taactatgcg ctgatcccaa cggcagaagt tccgctgact aacctggtgc gcggtgaaat
     3061 catcgatgaa gatgatctgc caattaagat gaccgcccac accccatgct tccgttctga
     3121 agccggttca tatggtcgtg acacccgtgg tctgatccgt atgcaccagt tcgacaaagt
     3181 tgaaatggtg cagatcgtgc gcccagaaga ctcaatggcg gcgctggaag agatgactgg
     3241 tcatgcagaa aaagtcctgc agttgctggg cctgccgtac cgtaaaatca tcctttgcac
     3301 tggcgacatg ggctttggcg cttgcaaaac ttacgacctg gaagtatgga tcccggcaca
     3361 gaacacctac cgtgagatct cttcctgctc caacgtttgg gatttccagg cacgtcgtat
     3421 gcaggcacgt tgccgcagca agtcggacaa gaaaacccgt ctggttcata ccctgaacgg
     3481 ttctggtctg gctgttggtc gtacgctggt tgcagtaatg gaaaactatc agcaggctga
     3541 tggtcgtatt gaagtaccag aagttctgcg tccgtatatg aacggactgg aatatattgg
     3601 cctcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     3661 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     3721 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     3781 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3841 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3901 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3961 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     4021 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4081 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     4141 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4201 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     4261 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4321 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4381 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4441 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4501 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4561 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4621 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4681 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4741 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     4801 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     4861 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     4921 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4981 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5041 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5101 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5161 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5221 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5281 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5341 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5401 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5461 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5521 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5581 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5641 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5701 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5761 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5821 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5881 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5941 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6001 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6061 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6121 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     6181 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     6241 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     6301 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     6361 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     6421 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     6481 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     6541 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     6601 gatgcctccg tgtaaggggg atttctgttc
//
