LOCUS       sequence-224906-        6232 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 6232)
  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 6232)
  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..6232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(113..130)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(284..872)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(364..383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1043..1834,1835..1903))
                     /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     1666..1685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1904..2008)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2035..2490)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2172..2193)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2384..2403
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2527..2574)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2570..2588
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2641..2658)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3533..3555
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3570..3594
                     /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(3594..3613)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3595..3613)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3701..3720)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3926..4003
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4004..5086
                     /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(4023..4042)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5895..6086
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6080..6102)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    join(6188..6232,1..98)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatatg cggtgtgaaa
       61 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
      121 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
      181 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
      241 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
      301 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
      361 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
      421 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
      481 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
      541 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
      601 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
      661 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
      721 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
      781 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
      841 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
      901 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
      961 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     1021 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     1081 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     1141 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     1201 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     1261 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     1321 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     1381 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     1441 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     1501 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     1561 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     1621 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     1681 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     1741 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     1801 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     1861 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     1921 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     1981 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaaattg
     2041 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     2101 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     2161 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2221 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2281 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     2341 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     2401 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     2461 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc aatccggata tagttcctcc
     2521 tttcagcaaa aaacccctca agacccgttt agaggcccca aggggttatg ctagttattg
     2581 ctcagcggtg gcagcagcca actcagcttc ctttcgggct ttgttagcag ccggatctca
     2641 gtggtggtgg tggtggtgct cgagaacaga accggagatg aagaaccact tgtcaataga
     2701 cttgtcaatt ggagcatctg cctttggaga agctggtggg atggcatcag aggcagcctt
     2761 ggagtaggtt ggggtgtcag gcaaagtaac attggtcaaa tcaataccct tgctgtcaga
     2821 agacttacca gcaattaatt gtttccagga gtcatgtgtt tccttgatga tatccaaagc
     2881 gtacttcttg ttcttagctt caccggagaa ggcaaattgg ttttctggct taccatctgg
     2941 gattttgtaa attctgaacc attcgttagt agccctcaac agacctggga agtatttctc
     3001 aacatcctca atgtcgttca attttggggc taatggatcg ttaatatcaa tggcaataac
     3061 tttccaatcg gtctcacctt catccaataa agccatgata cctagagcct taacttgctt
     3121 gacttgacca gtgtaagcaa tagtttcacc aatttccaac acatcaattg gatcgttgtc
     3181 accaactgcc ttagtttctg ggtggcttac gtttgggtct tcccaagttt gtgggaaagc
     3241 accatagttg tgaatgtaac catggtgagg gaaacagttt ctaacaaatc tcaacttgcc
     3301 cttcttggtg tcttggatga ttgggttcaa agtttcttcc ttggtgattt ctaacttggc
     3361 gttggtccaa cgtggaattt caacaaccat gttgaaaatg ttgttttcct tgtcagcgta
     3421 caagggaatg tcgtggaagg cagaaactgg cttaccatcc ttttcgatat aaactttgta
     3481 ttccaaggtg ttcttggcac caatttgtct ggtagtgtag gtcatatgta tatctccttc
     3541 ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata
     3601 gtgagtcgta ttaatttcgc gggatcgaga tcgatctcga tcctctacgc cggacgcatc
     3661 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     3721 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     3781 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     3841 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     3901 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat
     3961 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     4021 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     4081 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     4141 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     4201 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     4261 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     4321 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     4381 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     4441 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     4501 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     4561 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     4621 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     4681 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     4741 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     4801 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     4861 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     4921 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     4981 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     5041 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     5101 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca
     5161 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     5221 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg
     5281 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct
     5341 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc
     5401 aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga
     5461 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg
     5521 agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg
     5581 tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta
     5641 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat
     5701 taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg
     5761 ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta
     5821 tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt
     5881 acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca
     5941 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag
     6001 acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt
     6061 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     6121 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     6181 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact gg
//
