LOCUS       sequence-258523-        6308 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 6308)
  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 6308)
  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-MTF-His
FEATURES             Location/Qualifiers
     source          1..6308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    26..165
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     81..100
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(180..197)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(351..939)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(431..450)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1110..1901,1902..1970))
                     /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     1733..1752
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1971..2075)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2101..2556)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2238..2259)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2450..2469
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2593..2640)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2636..2654
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2707..2724)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3683..3705
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3720..3744
                     /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(3744..3763)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3745..3763)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3847..3866)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4072..4149
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4150..5232
                     /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(4169..4188)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6041..6232
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6226..6248)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
       61 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg
      121 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
      181 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
      241 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
      301 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
      361 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
      421 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
      481 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
      541 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
      601 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
      661 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
      721 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
      781 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
      841 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
      901 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
      961 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     1021 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     1081 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     1141 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     1201 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     1261 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     1321 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     1381 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg
     1441 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     1501 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     1561 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     1621 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     1681 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     1741 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     1801 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     1861 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     1921 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     1981 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     2041 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     2101 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     2161 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     2221 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     2281 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     2341 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     2401 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     2461 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     2521 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc cggatatagt
     2581 tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag
     2641 ttattgctca gcggtggcag cagccaactc agcttccttt cgggctttgt tagcagccgg
     2701 atctcagtgg tggtggtggt ggtgctcgag gaccagacgg ttgcccggaa caaaccattc
     2761 ccgacgagag ttcaggaggt cttgcgcgct catcgctttc ttacccgcag gttgtaacga
     2821 gagcaggttc aggatgccat caccagtcgc aacctgaatg ccttgtttgt tggcttcaag
     2881 gatcgttcct ggtgcagcgt tggttgccgt atcaatgacc gatgctttcc agactttaac
     2941 cggctgtcct tcaatttcca gccagctcat tggccatgga ttgaaagcgc gaatgcagcg
     3001 ttcaagctgt gctgccgaaa gtgaccagtc aatacgcgct tcttctttac tcaacttctc
     3061 ggcgtaagtg acaagagttt cgtcctgaac ttctggtttc gccgtgccgt ctgccagttg
     3121 tttcaacgtg gtgataagcc cttgtgggcc aagctctgcc agcttgtcgt acagcgtacc
     3181 actggtatct tctgcagtaa tcgggcagga gagcttatag agcatatcac cggtgtctaa
     3241 accgacatcc atttgcataa tggtcacacc agtttctgca tcacccgccc atagtgagcg
     3301 ttggattggt gcagcaccgc gccagcgtgg cagcagtgaa ccatgaacgt tgatacagcc
     3361 aagacgcggc atctccagca ctgctttcgg cagaattaaa ccataggcga cgacgaccat
     3421 aacatcagcc tgcagttcgg cgaccagttg ctggttttct tgtggacgca gggaaacagg
     3481 ttgaaaaacg ggcagacctt tttcctcagc cagaacttta accgggctgg gcatcagttt
     3541 tttaccgcgt cctgccggtc ggtctggctg ggtgaacacg ccaacgacgt tatgaccaga
     3601 agacaacagc gcgtcgagat gacgcgctgc aaagtcaggt gtacccgcaa aaataatacg
     3661 tagtgattct gacatatgta tatctccttc ttaaagttaa acaaaattat ttctagaggg
     3721 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
     3781 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
     3841 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     3901 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     3961 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     4021 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc
     4081 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     4141 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     4201 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     4261 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     4321 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     4381 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     4441 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     4501 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     4561 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     4621 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     4681 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     4741 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     4801 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     4861 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     4921 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     4981 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     5041 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     5101 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     5161 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     5221 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc
     5281 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     5341 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     5401 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     5461 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     5521 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt
     5581 gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg
     5641 ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc
     5701 tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac
     5761 gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct
     5821 accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt
     5881 tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg
     5941 ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat
     6001 tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac
     6061 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa
     6121 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga
     6181 gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     6241 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     6301 gggcgcgt
//
