LOCUS       sequence-454219-        6650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  6xHis-PSP-GS-PANN(75-150)-(5aaGS)-Msp1(36-362)_W166A/Y167A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6650)
  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 6650)
  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: pAM248
FEATURES             Location/Qualifiers
     source          1..6650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     131..150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     384..401
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    416..555
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(481..500)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     641..663
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(657..848)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1420..2502)
                     /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     2464..2483
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2503..2580)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2786..2805
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2893..2911
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2912..2936
                     /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             2951..2973
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2992..3009
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3022..3045
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        4356..4374
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4375..4399
                     /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)."
     CDS             4508..4552
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4590..4608)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4604..4651
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4688..5143
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4775..4794)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4985..5006
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5258..6049,6050..6118))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGREQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5881..5900
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6119..6211)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6292..6650,1..230)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
       61 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      121 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      181 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      241 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
      301 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
      361 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
      421 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
      481 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      541 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      601 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
      661 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
      721 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
      781 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
      841 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
      901 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
      961 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     1021 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     1081 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     1141 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     1201 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     1261 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgctagt catgccccgc
     1321 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     1381 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     1441 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1501 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     1561 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     1621 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     1681 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     1741 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     1801 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     1861 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     1921 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     1981 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     2041 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     2101 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     2161 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     2221 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     2281 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     2341 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     2401 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     2461 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     2521 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     2581 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     2641 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     2701 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     2761 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     2821 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg
     2881 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     2941 tagaaataat tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcaccat
     3001 catcaccaca gccaggatcc gctggaagtt ctgtttcaag gcccgggtag caaggaaaat
     3061 gagattctgc gccgtgagct ggaccgtatg cgtgtgccgc cgctgattgt tggtaccgtg
     3121 gttgataaag tgggcgagcg taaggtggtt gtgaaaagca gcaccggtcc gagcttcctg
     3181 gttaacgtga gccactttgt taacccggat gatctggcgc cgggcaagcg tgtgtgcctg
     3241 aaccagcaaa ccctgaccgt tgtggacgtt ctgccggaac tggaaggtag cggcagcggt
     3301 agcggtccgc tgagcggtaa aagccgtgaa agcaaggcga aacagagcct gcaatgggag
     3361 aagctggtta aacgtagccc ggcgctggcg gaagtgaccc tggatgcgta cgaacgtacc
     3421 atcctgagca gcattgtgac cccggacgag atcaacatta ccttccagga catcggtggt
     3481 ctggacccgc tgattagcga cctgcacgaa agcgttatct acccgctgat gatgccggaa
     3541 gtgtatagca acagcccgct gctgcaagcg ccgagcggtg ttctgctgta cggtccgccg
     3601 ggttgcggca aaaccatgct ggcgaaggcg ctggcgaaag aaagcggtgc gaacttcatc
     3661 agcattcgta tgagcagcat catggataag gcggcgggcg agagcaacaa aattgtggac
     3721 gcgatgttta gcctggcgaa caagctgcag ccgtgcatca ttttcatcga cgagattgat
     3781 agctttctgc gtgaacgtag cagcaccgat cacgaggtta ccgcgaccct gaaagcggaa
     3841 tttatgaccc tgtgggacgg tctgctgaac aacggccgtg tgatgatcat tggtgcgacc
     3901 aaccgtatca acgatattga cgatgcgttc ctgcgtcgtc tgccgaagcg ttttctggtt
     3961 agcctgccgg gcagcgatca gcgttacaaa atcctgagcg tgctgctgaa ggacaccaaa
     4021 ctggacgagg atgaattcga tctgcaactg attgcggaca acaccaaggg ttttagcggc
     4081 agcgatctga aagaactgtg ccgtgaagcg gcgctggatg cggcgaagga gtatatcaag
     4141 cagaaacgtc aactgatcga cagcggtacc attgacgtta acgataccag cagcctgaaa
     4201 attcgtccgc tgaagaccaa agatttcacc aaaaaactgc gtatggatgc gaccagcacc
     4261 ctgagcagcc aaccgctgga ctaaaagctt gcggccgcat aatgcttaag tcgaacagaa
     4321 agtaatcgta ttgtacacgg ccgcataatc gaaattaata cgactcacta taggggaatt
     4381 gtgagcggat aacaattccc catcttagta tattagttaa gtataagaag gagatataca
     4441 tatggcagat ctcaattgga tatcggccgg ccacgcgatc gctgacgtcg gtaccctcga
     4501 gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact cgtctactag
     4561 cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     4621 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     4681 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4741 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4801 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4861 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4921 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4981 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5041 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5101 aaatttaacg cgaattttaa caaaatatta acgtttacaa tttctggcgg cacgatggca
     5161 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5221 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5281 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5341 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5401 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5461 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5521 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5581 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5641 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5701 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5761 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5821 agtcattctg agaatagtgt atgcggcgac cgagttgctc tcgcccggcg tcaatacggg
     5881 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5941 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6001 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6061 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6121 tcttcctttt tcaatcatga ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6181 atatttgaat gtatttagaa aaataaacaa ataggtcatg accaaaatcc cttaacgtga
     6241 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6301 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6361 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6421 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6481 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6541 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6601 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
//
