LOCUS       sequence-429652-        7235 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses hisMBP-tagged rat oxidation-resistant RNase inhibitor in 
            E. coli hosts.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7235)
  TITLE     Bacterial expression plasmids for production of recombinant PNK
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7235)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pET-hisMBP-rRNaseI
FEATURES             Location/Qualifiers
     source          1..7235
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(404..451)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     447..465
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1887..1907)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1929..1940)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(1989..3089)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(1992..2015)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      2123..2132
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(3093..3110)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(3120..3137)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3157..3179
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(3210..3229)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3211..3229)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3313..3332)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             4790..4981
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4975..4997)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5083..5222
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5138..5157
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5237..5254)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5408..5996)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5488..5507)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6167..6958,6959..7027))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6790..6809
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7028..7132)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7200..7218
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata
      241 tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg
      301 ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac
      361 cacacccgtc ctgtggatat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
      421 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact
      481 cagcttcctt tcgggctttg ttagcagccg gatccttagc taataatgcg cagggacggg
      541 cgttcttctt ccagggcgcg cagctgatct tccacttcat cggtccagta gatatcgtac
      601 agcaccagct gctgtaaaat gcagctcggc tgtttcaggc tttccagcag ttgcaacacg
      661 ccgttatcgc ccatacagtt gtttgacaga tctaattcgc gcagcgaacg gttcgccagc
      721 agaacggtcg ccaggctgct gcaacccgaa tcggtcacat cgcaatcgcc caaccacagc
      781 actctcagaa cggtatccgg ataacccagc gctttacata attccacaac gccagaatca
      841 cccagcgggt tcgacgacat ctgcagttca aacagtgagc tgtttttggt cagcacgctg
      901 caaaaatgcg ggcagctggc ggcggtcagg ctgcaggttt tcacccacag cgattccaac
      961 tggcaacccg gttccaataa ggattcacac agcagctgcg cgccttcatc tttcagttca
     1021 ttgcccgcca ggctcagttc tttcaggctc tgtttcgcac gcaggacacg gcacaggtct
     1081 ttacagcctt cggcggtcac gtcacaatcc cacagccaca gggtgcgcag gcgacaggac
     1141 ggcagcagca ggccagagca taaagcggcg atgcccgtgt tgcccagttt gttactgccc
     1201 agatccagtt cctgcaggct cgctttgctc gcgacaacat cgcacagatc tttgcaattc
     1261 gcggaggtaa taccacaatt ttccagtttc agagactcca gctgacacgc gctatctttc
     1321 agaccctggc acagggtgtg gatgcctgct tcatgaaagt cgttgttgct cagcaccagt
     1381 tctttaaaat ccggtttaac gcgcagcacg cttgccagcg gttcgcagct ggtcgcggtc
     1441 agattgcaat attccagctg cagtttttcc agacggcact gcggatcgcg taagccttcg
     1501 cacagcagtt tcaggccttc atcgcccagc ggattatcgt tcagatgcag ttcacgcagc
     1561 gtacttaagc tacgcagcac atccggtaag acgccacagc ccgcttccgt caggctacag
     1621 ttctgcagtg acagtttctg aattttacac gtcgggttct ggaggccctg caggaccagg
     1681 ccaacgcccg catcgccaag ttcgttggta cgcagactca gttccgtcag cgccgggttc
     1741 gcctgaatgg cgctacggat atctttgcag cgcacttcgg tcagaccgca atcatccagg
     1801 cgcaccacct gatactgctg gatcagcggc agcagttcgg tccagcgggc atcgctcagc
     1861 tgttcgcact gaatatccag gctcatgccc tggaaataca ggttctcgct agcgaattcg
     1921 gatcctcccc ttccctcgat cccgaggttg ttgttattgt tattgttgtt gttgttcgag
     1981 ctcgaattag tctgcgcgtc tttcagggct tcatcgacag tctgacgacc gctggcggcg
     2041 ttgatcaccg cagtacgcac ggcataccag aaagcggaca tctgcgggat gttcggcatg
     2101 atttcacctt tctgggcgtt ttccatggtg gcggcaatac gtggatcttt cgccaactct
     2161 tcctcgtaag acttcagcgc tacggcaccc agcggtttgt ctttattaac cgcttccaga
     2221 ccttcatcag tcagcagata gttttcgagg aactcttttg ccagctcttt gttcggactg
     2281 gcggcgttaa tacctgcgct cagcacgcca acgaacggtt tggatggttg acccttgaag
     2341 gtcggcagta ccgttacacc ataattcact ttgctggtgt cgatgttgga ccatgcccac
     2401 gggccgttga tggtcatcgc tgtttcgcct ttattaaagg cagcttctgc gatggagtaa
     2461 tcggtgtctg cattcatgtg tttgttttta atcaggtcaa ccaggaaggt cagacccgct
     2521 ttcgcgccag cgttatccac gcccacgtct ttaatgtcgt acttgccgtt ttcatacttg
     2581 aacgcataac ccccgtcagc agcaatcagc ggccaggtga agtacggttc ttgcaggttg
     2641 aacatcagcg cgctcttacc tttcgctttc agttctttat ccagcgccgg gatctcttcc
     2701 caggtttttg gcgggttcgg cagcagatct ttgttataaa tcagcgataa cgcttcaaca
     2761 gcgatcgggt aagcaatcag cttgccgttg taacgtacgg catcccaggt aaacggatac
     2821 agcttgtcct ggaacgcttt gtccggggtg atttcagcca acaggccaga ttgagcgtag
     2881 ccaccaaagc ggtcgtgtgc ccagaagata atgtcagggc catcgccagt tgccgcaacc
     2941 tgtgggaatt tctcttccag tttatccgga tgctcaacgg tgactttaat tccggtatct
     3001 ttctcgaatt tcttaccgac ttcagcgaga ccgttatagc ctttatcgcc gttaatccag
     3061 attaccagtt taccttcttc gattttcata tggctgccgc gcggcaccag gccgctgctg
     3121 tgatgatgat gatgatggct gctgcccata tgtatatctc cttcttaaag ttaaacaaaa
     3181 ttatttctag agggaaaccg ttgtggtctc cctatagtga gtcgtattaa tttcgcggga
     3241 tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg
     3301 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
     3361 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
     3421 tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
     3481 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga ccgatgccct
     3541 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
     3601 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg
     3661 tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg
     3721 tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt
     3781 tcggcgagaa gcaggccatt atcgccggca tggcggccga cgcgctgggc tacgtcttgc
     3841 tggcgttcgc gacgcgaggc tggatggcct tccccattat gattcttctc gcttccggcg
     3901 gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac gaccatcagg
     3961 gacagcttca aggatcgctc gcggctctta ccagcctaac ttcgatcatt ggaccgctga
     4021 tcgtcacggc gatttatgcc gcctcggcga gcacatggaa cgggttggca tggattgtag
     4081 gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca
     4141 cctcgacctg aatggaagcc ggcggcacct cgctaacgga ttcaccactc caagaattgg
     4201 agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc agaacatatc
     4261 catcgcgtcc gccatctcca gcagccgcac gcggcgcatc tcgggcagcg ttgggtcctg
     4321 gccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg
     4381 ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg
     4441 caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag
     4501 tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg
     4561 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg
     4621 agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc
     4681 cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat
     4741 cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca
     4801 ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga
     4861 gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca
     4921 cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     4981 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     5041 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     5101 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     5161 agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     5221 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5281 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5341 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5401 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5461 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5521 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5581 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5641 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5701 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5761 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5821 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     5881 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5941 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6001 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6061 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6121 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6181 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6241 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6301 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6361 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6421 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6481 ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6541 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6601 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6661 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6721 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6781 caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6841 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6901 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6961 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7021 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7081 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7141 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     7201 ataggcgtat cacgaggccc tttcgtcttc aagaa
//
