LOCUS       sequence-451035-        6908 bp    DNA     circular SYN 15-APR-2025
DEFINITION  6xHis-PSP-GS-Hcp1(1-162)-(6aaSG)-Msp1(36-362).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6908)
  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 6908)
  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"; 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: pAM247
FEATURES             Location/Qualifiers
     source          1..6908
                     /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        4614..4632
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4633..4657
                     /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             4766..4810
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4848..4866)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4862..4909
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4946..5401
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5033..5052)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5243..5264
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5516..6307,6308..6376))
                     /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     6139..6158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6377..6469)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6550..6908,1..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 gtccaggatc aatggctgtt
     3061 gatatgttca tcaagatcgg cgacgtcaag ggtgagtcca aggacaagac tcacgccgag
     3121 gaaatcgacg tgctggcatg gagctggggc atgtcccagt ccgggtcgat gcacatgggc
     3181 ggtggcggcg gcgccggcaa ggtcaacgtg caggacctgt cgttcaccaa gtacatcgac
     3241 aagtccacgc ccaacctgat gatggcctgc tccagcggca agcactatcc gcaggcgaag
     3301 ctgaccatcc gcaaggccgg cggcgagaac caggtcgagt acctgatcat caccctgaag
     3361 gaagtcctgg tgtcctcggt gagcaccggc ggcagcggtg gcgaggatcg cctgaccgag
     3421 aacgtgaccc tgaacttcgc ccaggtccag gtcgactacc agccgcagaa ggcggatggc
     3481 gcgaaggacg gcggtccggt caagtacggc tggaacatcc gccagaacgt gcaggccgga
     3541 tcagggtctg gtagcggctc aggcccgctt tccggtaaat cgcgtgagag caaggcgaaa
     3601 cagagcctgc aatgggagaa gctggttaaa cgtagcccgg cgctggcgga agtgaccctg
     3661 gatgcgtacg aacgtaccat cctgagcagc attgtgaccc cggacgagat caacattacc
     3721 ttccaggaca tcggtggtct ggacccgctg attagcgacc tgcacgaaag cgttatctac
     3781 ccgctgatga tgccggaagt gtatagcaac agcccgctgc tgcaagcgcc gagcggtgtt
     3841 ctgctgtacg gtccgccggg ttgcggcaaa accatgctgg cgaaggcgct ggcgaaagaa
     3901 agcggtgcga acttcatcag cattcgtatg agcagcatca tggataagtg gtatggcgag
     3961 agcaacaaaa ttgtggacgc gatgtttagc ctggcgaaca agctgcagcc gtgcatcatt
     4021 ttcatcgacg agattgatag ctttctgcgt gaacgtagca gcaccgatca cgaggttacc
     4081 gcgaccctga aagcggaatt tatgaccctg tgggacggtc tgctgaacaa cggccgtgtg
     4141 atgatcattg gtgcgaccaa ccgtatcaac gatattgacg atgcgttcct gcgtcgtctg
     4201 ccgaagcgtt ttctggttag cctgccgggc agcgatcagc gttacaaaat cctgagcgtg
     4261 ctgctgaagg acaccaaact ggacgaggat gaattcgatc tgcaactgat tgcggacaac
     4321 accaagggtt ttagcggcag cgatctgaaa gaactgtgcc gtgaagcggc gctggatgcg
     4381 gcgaaggagt atatcaagca gaaacgtcaa ctgatcgaca gcggtaccat tgacgttaac
     4441 gataccagca gcctgaaaat tcgtccgctg aagaccaaag atttcaccaa aaaactgcgt
     4501 atggatgcga ccagcaccct gagcagccaa ccgctggact aaaagcttgc ggccgcataa
     4561 tgcttaagtc gaacagaaag taatcgtatt gtacacggcc gcataatcga aattaatacg
     4621 actcactata ggggaattgt gagcggataa caattcccca tcttagtata ttagttaagt
     4681 ataagaagga gatatacata tggcagatct caattggata tcggccggcc acgcgatcgc
     4741 tgacgtcggt accctcgagt ctggtaaaga aaccgctgct gcgaaatttg aacgccagca
     4801 catggactcg tctactagcg cagcttaatt aacctaggct gctgccaccg ctgagcaata
     4861 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     4921 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4981 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     5041 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5101 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5161 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5221 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     5281 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5341 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5401 tctggcggca cgatggcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5461 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5521 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5581 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5641 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5701 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5761 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5821 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5881 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5941 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6001 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6061 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6121 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6181 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6241 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6301 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6361 aatgttgaat actcatactc ttcctttttc aatcatgatt gaagcattta tcagggttat
     6421 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggtcatgac
     6481 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6541 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6601 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6661 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     6721 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6781 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6841 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6901 gcgaacga
//
