LOCUS       sequence-120514-        6769 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression vector MBP/hexahistidine tagged variant of the MS2 coat 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6769)
  AUTHORS   Batey RT, Kieft JS
  TITLE     Improved native affinity purification of RNA.
  JOURNAL   RNA. 2007 Aug;13(8):1384-9. Epub 2007 Jun 4.
  PUBMED    17548432
REFERENCE   2  (bases 1 to 6769)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "RNA."; 
            date: "2007-08"; volume: "13(8)"; pages: "1384-9. Epub 2007 Jun 4"
COMMENT     Sequence Label: pHMM
FEATURES             Location/Qualifiers
     source          1..6769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(528..539)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(570..1667)
                     /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="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(573..596)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      704..713
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1668..1691)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             1702..1724
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1739..1763
                     /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(1763..1782)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1764..1782)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1870..1889)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2095..2172
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2173..3255
                     /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(2192..2211)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4064..4255
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4249..4271)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4357..4499
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4412..4431
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4514..4531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4685..5273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4765..4784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5384..5403)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5395..6210
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5395..6210
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5468..5487)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6303..6758)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6440..6461)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6652..6671
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttag
      181 tagatgccgg agtttgctgc gattgctgag ggaatcgggt ttccatcttt taggagacct
      241 tgcattgcct taacaataag ctcgcagtcg gaattcgtgg cgaaaattgg aatggttagt
      301 tccatattta agtacgaacg ccaggcgcct ttaggcacct cgactttgat ggtgtatttg
      361 cgattctgcg cagagctctg acgaacgcta caggttactt tgtaagcctg tgaacgcgag
      421 ttagagctga tccattcagc gatcccgtta gcgaagttgc ttggggcgac agtcacgtcg
      481 ccagttccgc cattgtcgac gagaacgaac tgagtaaagt tagaagccct accctcgatg
      541 gatccccggc cgggtaccga gctcgaatta gtctgcgcgt ctttcagggc ttcatcgaca
      601 gtctgacgac cgctggcggc gttgatcacc gcagtacgca cggcatacca gaaagcggac
      661 atctgcggga tgttcggcat gatttcacct ttctgggcgt tttccatggt ggcggcaata
      721 cgtggatctt tcgccaactc ttcctcgtaa gacttcagcg ctacggcacc cagcggtttg
      781 tctttattaa ccgcttccag accttcatca gtcagcagat agttttcgag gaactctttc
      841 gccagctctt tgttcggact ggcggcgtta atacctgcgc tcagcacgcc aacgaacggt
      901 ttggatggtt gacccttgaa ggtcggcagt accgttacac cataattcac tttgctggtg
      961 tcgatgttgg accatgccca cgggccgttg atggtcatcg ctgtttcgcc tttattaaag
     1021 gcagcttctg cgatggagta atcggtgtct gcattcatgt gtttgttttt aatcaggtca
     1081 accaggaagg tcagacccgc tttcgcgcca gcgttatcca cgcccacgtc tttaatgtcg
     1141 tacttgccgt tttcatactt gaacgcataa cccccgtcag cagcaatcag cggccaggtg
     1201 aagtacggtt cttgcaggtt gaacatcagc gcgctcttac ctttcgcttt cagttcttta
     1261 tccagcgccg ggatctcttc ccaggttttt ggcgggttcg gcagcagatc tttgttataa
     1321 atcagcgata acgcttcaac agcgatcggg taagcaatca gcttgccgtt gtaacgtacg
     1381 gcatcccagg taaacggata cagcttgtcc tggaacgctt tgtccggggt gatttcagcc
     1441 aacaggccag attgagcgta gccaccaaag cggtcgtgtg cccagaagat aatgtcaggg
     1501 ccatcgccag ttgccgcaac ctgtgggaat ttctcttcca gtttatccgg atgctcaacg
     1561 gtgactttaa ttccggtatc tttctcgaat ttcttaccga cttcagcgag accgttatag
     1621 cctttatcgc cgttaatcca gattaccagt ttaccttctt cgattttgtg atggtgatgg
     1681 tgatggtgat gcatatgtat atctccttct taaagttaaa caaaattatt tctagagggg
     1741 aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat
     1801 cgatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
     1861 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
     1921 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
     1981 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     2041 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
     2101 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     2161 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     2221 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     2281 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     2341 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     2401 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     2461 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     2521 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     2581 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     2641 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     2701 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     2761 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     2821 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     2881 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     2941 attaccgagt ccgggctgcg cgttggtgcg gacatctcgg tagtgggata cgacgatacc
     3001 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     3061 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     3121 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     3181 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     3241 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
     3301 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     3361 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     3421 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
     3481 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
     3541 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg
     3601 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     3661 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     3721 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     3781 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     3841 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     3901 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     3961 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     4021 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     4081 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     4141 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     4201 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     4261 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     4321 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     4381 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     4441 caccatatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     4501 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4561 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4621 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4681 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4741 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4801 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4861 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4921 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4981 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5041 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5101 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     5161 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5221 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5281 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5341 tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     5401 catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat
     5461 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga
     5521 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     5581 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     5641 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     5701 gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     5761 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     5821 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     5881 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     5941 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     6001 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     6061 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     6121 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     6181 tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga
     6241 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6301 tgaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     6361 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     6421 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     6481 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     6541 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     6601 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     6661 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     6721 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
