LOCUS       sequence-451052-        7441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  6xHis-PSP-MBP-Pex3(40-441).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7441)
  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 7441)
  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: pAM257
FEATURES             Location/Qualifiers
     source          1..7441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(490..1572)
                     /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     1534..1553
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1573..1650)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1856..1875
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1963..1982
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1963..1981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1982..2006
                     /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             2021..2043
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2062..2079
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2086..2109
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2110..3210
                     /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     3184..3207
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     primer_bind     complement(4451..4469)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4465..4512
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4549..5004
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4636..4655)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4846..4867
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5119..5910,5911..5979))
                     /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     5742..5761
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5980..6072)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6153..6741
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6642..6661
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6895..6912
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6927..7066
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6992..7011)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7152..7174
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7168..7359)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
       61 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
      121 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
      181 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
      241 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
      301 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
      361 aggagcacga tcatgctagt catgccccgc gcccaccgga aggagctgac tgggttgaag
      421 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
      481 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
      541 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
      601 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
      661 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
      721 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
      781 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
      841 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
      901 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
      961 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     1021 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     1081 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     1141 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     1201 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     1261 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     1321 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     1381 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     1441 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     1501 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     1561 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     1621 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     1681 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     1741 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     1801 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     1861 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     1921 gtccggcgta gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag
     1981 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     2041 agatatacca tgggcagcag ccatcaccat catcaccaca gccagctgga agttctgttt
     2101 caaggcccga tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
     2161 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
     2221 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
     2281 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
     2341 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
     2401 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
     2461 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
     2521 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
     2581 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
     2641 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
     2701 gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc
     2761 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
     2821 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
     2881 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     2941 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     3001 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     3061 attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     3121 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     3181 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgaagagatg gttgtataaa
     3241 cagcagttac ggattactga gcaacacttc atcaaagaac agatcaaaag aaggttcgag
     3301 cagacacagg aagactcgtt gtacacaata tacgagctac ttcctgtatg gagaatggtt
     3361 ttgaatgaaa acgatttgaa tttggacagc atcgttaccc aattaaagga ccaaaagaac
     3421 cagttgacta gagcaaagtc tagtgaaagc agagaaagtt cgccgttaaa aagtaaagct
     3481 gagttgtgga acgagttaga actaaagagt ttgatcaagc tggtgacagt gacgtacaca
     3541 gtatcgtcgc tgattctttt aacaagactg caactaaata tcctgacaag aaacgagtac
     3601 ctggactccg caataaaatt aaccatgcag caggaaaact gcaacaaact acagaatagg
     3661 ttctataact gggttacatc gtggtggagc gatccggagg acaaagcgga tgatgcaatg
     3721 gtaatggcag caaaaaagtc aaagaaagaa ggccaggaag tgtatattaa tgagcaagct
     3781 ttcctatcac tatcatggtg gattcttaac aaggggtggt tgagttacaa tgaaataatt
     3841 acaaatcaaa tcgaaattga atttgacggc atacacccaa gagatactct aacgttagaa
     3901 gaatttagtt ctcgtctcac gaacatattc cggaacacga attcccaaat attccagcaa
     3961 aataataata acctcacgtc tattttgctt cctaaagatt ccagtggaca ggagtttctt
     4021 ctgtcacaga cgcttgacgc ggatgcttta acaagttttc actctaacac actagttttt
     4081 aatcaattgg tgaatgaact gacccaatgt atcgaaagca cagcaacatc catagtgtta
     4141 gagtctctaa taaatgaatc attccatttc ataatgaaca aggtcggcat aaagacaata
     4201 gccaagaaaa aacctggcca agaagatcag caacagtatc aaatggcagt tttcgctatg
     4261 tcgatgaaag actgttgcca agaaatgctt caaacaactg ccgggtcttc ccatagcggc
     4321 agcgtgaacg aatacctggc cactctggat tctgtgcagc cgctcgatga tctgagcgcc
     4381 agcgtataca gcaactttgg cgtctccagc tcgttttcct tcaagcctta attaacctag
     4441 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     4501 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     4561 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4621 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tcttgccacg ttcgccggct
     4681 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4741 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     4801 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4861 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4921 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4981 acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat
     5041 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5101 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5161 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5221 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5281 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5341 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     5401 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     5461 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     5521 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     5581 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     5641 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     5701 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5761 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5821 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5881 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5941 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
     6001 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6061 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6121 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6181 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6241 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     6301 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6361 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6421 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6481 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6541 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6601 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6661 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6721 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6781 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6841 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     6901 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     6961 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     7021 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     7081 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     7141 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     7201 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     7261 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     7321 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     7381 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     7441 t
//
