LOCUS       sequence-229750-        6228 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for Inorganic pyrophosphatase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6228)
  AUTHORS   Lavickova B, Maerkl SJ
  TITLE     A simple, robust, and low-cost method to produce the PURE cell-free 
            system.
  JOURNAL   ACS Synth Biol. 2019 Jan 11. doi: 10.1021/acssynbio.8b00427.
  PUBMED    30632751
REFERENCE   2  (bases 1 to 6228)
  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"; journalName: "ACS Synth 
            Biol. 2019 Jan 11. doi: 10.1021/acssynbio.8b00427."
COMMENT     Sequence Label: pET-21a(+)-IPP1-His
FEATURES             Location/Qualifiers
     source          1..6228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1292..1311
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1399..1418
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1399..1417
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1418..1442
                     /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             1457..1479
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2354..2371
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2424..2442)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2438..2485
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2522..2977
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2609..2628)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2819..2840
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3003..3107
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3108..3176,3177..3968)
                     /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(3326..3345)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4139..4727
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4628..4647
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4881..4898
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4913..5052
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4978..4997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5138..5160
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5154..5345)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6154..6228,1..1008))
                     /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"
ORIGIN
        1 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1201 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1261 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     1321 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg
     1381 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     1441 cccctctaga aataattttg tttaacttta agaaggagat atacatatga cctacactac
     1501 cagacaaatt ggtgccaaga acaccttgga atacaaagtt tatatcgaaa aggatggtaa
     1561 gccagtttct gccttccacg acattccctt gtacgctgac aaggaaaaca acattttcaa
     1621 catggttgtt gaaattccac gttggaccaa cgccaagtta gaaatcacca aggaagaaac
     1681 tttgaaccca atcatccaag acaccaagaa gggcaagttg agatttgtta gaaactgttt
     1741 ccctcaccat ggttacattc acaactatgg tgctttccca caaacttggg aagacccaaa
     1801 cgtaagccac ccagaaacta aggcagttgg tgacaacgat ccaattgatg tgttggaaat
     1861 tggtgaaact attgcttaca ctggtcaagt caagcaagtt aaggctctag gtatcatggc
     1921 tttattggat gaaggtgaga ccgattggaa agttattgcc attgatatta acgatccatt
     1981 agccccaaaa ttgaacgaca ttgaggatgt tgagaaatac ttcccaggtc tgttgagggc
     2041 tactaacgaa tggttcagaa tttacaaaat cccagatggt aagccagaaa accaatttgc
     2101 cttctccggt gaagctaaga acaagaagta cgctttggat atcatcaagg aaacacatga
     2161 ctcctggaaa caattaattg ctggtaagtc ttctgacagc aagggtattg atttgaccaa
     2221 tgttactttg cctgacaccc caacctactc caaggctgcc tctgatgcca tcccaccagc
     2281 ttctccaaag gcagatgctc caattgacaa gtctattgac aagtggttct tcatctccgg
     2341 ttctgttctc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     2401 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     2461 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     2521 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2581 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2641 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2701 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2761 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2821 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2881 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2941 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     3001 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3061 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3121 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3181 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     3241 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     3301 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     3361 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     3421 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3481 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3541 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3601 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     3661 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3721 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     3781 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3841 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3901 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3961 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     4021 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4081 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4141 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4201 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4261 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4321 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4381 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4441 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4501 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4561 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4621 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4681 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4741 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4801 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4861 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4921 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     4981 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     5041 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5101 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5161 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     5221 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     5281 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     5341 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     5401 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     5461 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     5521 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     5581 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     5641 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     5701 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     5761 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     5821 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     5881 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     5941 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     6001 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     6061 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     6121 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6181 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggc
//
