LOCUS       sequence-451045-        6239 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  6xHis-PSP-MBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6239)
  AUTHORS   Castanzo DT, LaFrance B, Martin A
  TITLE     The AAA+ ATPase Msp1 is a processive protein translocase with robust
            unfoldase activity.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Jun 30;117(26):14970-14977. doi: 
            10.1073/pnas.1920109117. Epub 2020 Jun 15.
  PUBMED    32541053
REFERENCE   2  (bases 1 to 6239)
  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: 
            "10.1073/pnas.1920109117"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2020-06-30- 30"; volume: "117"; issue: "26"; pages: 
            "14970-14977"
COMMENT     Sequence Label: pAM258
FEATURES             Location/Qualifiers
     source          1..6239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..36
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(30..221)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(793..1875)
                     /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     1837..1856
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1876..1953)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2159..2178
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2266..2285
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2266..2284
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2285..2309
                     /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             2324..2346
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2365..2382
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2389..2412
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2413..3513
                     /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     3487..3510
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     primer_bind     complement(3552..3570)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3566..3613
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3650..4105
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3737..3756)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3947..3968
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4220..5011,5012..5080))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4843..4862
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5081..5173)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5254..5842
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5743..5762
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5996..6013
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6028..6167
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6093..6112)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
       61 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
      121 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
      181 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
      241 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
      301 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
      361 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
      421 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
      481 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
      541 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
      601 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
      661 gacaggagca cgatcatgct agtcatgccc cgcgcccacc ggaaggagct gactgggttg
      721 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
      781 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
      841 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
      901 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
      961 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     1021 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     1081 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     1141 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     1201 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     1261 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     1321 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     1381 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     1441 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     1501 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     1561 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     1621 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     1681 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     1741 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     1801 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     1861 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     1921 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     1981 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     2041 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     2101 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     2161 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     2221 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta
     2281 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
     2341 aggagatata ccatgggcag cagccatcac catcatcacc acagccagct ggaagttctg
     2401 tttcaaggcc cgatgaaaat cgaagaaggt aaactggtaa tctggattaa cggcgataaa
     2461 ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc
     2521 accgttgagc atccggataa actggaagag aaattcccac aggttgcggc aactggcgat
     2581 ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg
     2641 ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat
     2701 gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg
     2761 atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg
     2821 gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac
     2881 ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag
     2941 tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg
     3001 gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct
     3061 gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc
     3121 gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc
     3181 aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg
     3241 gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa
     3301 gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggc gaaagatcca
     3361 cgtattgccg ccactatgga aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag
     3421 atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag
     3481 actgtcgatg aagccctgaa agacgcgcag actaattcga gctcgtgatt aattaaccta
     3541 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     3601 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     3661 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3721 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttcttgccac gttcgccggc
     3781 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3841 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3901 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3961 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     4021 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4081 aacaaaatat taacgtttac aatttctggc ggcacgatgg catgagatta tcaaaaagga
     4141 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4201 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4261 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4321 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4381 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4441 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4501 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4561 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4621 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4681 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4741 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4801 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4861 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4921 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4981 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5041 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatcat
     5101 gattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5161 aaaaataaac aaataggtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5221 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5281 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5341 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5401 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5461 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5521 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5581 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5641 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5701 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5761 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5821 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5881 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5941 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     6001 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     6061 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6121 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     6181 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaa
//
