LOCUS       sequence-451050-        6329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mEOS3.2-MBP-Pex15(1-253)-PSP-FLAG-6xHis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6329)
  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 6329)
  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: pAM259
FEATURES             Location/Qualifiers
     source          1..6329
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(125..1207)
                     /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     1169..1188
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1208..1285)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1331..1350
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1331..1349
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1350..1374
                     /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             1418..2095
                     /codon_start=1
                     /product="improved monomeric variant of green-to-red 
                     photoswitchable fluorescent protein EosFP (Zhang et al., 
                     2012)"
                     /label=mEos3.2
                     /translation="MSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVK
                     EGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNAR
                     NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLL
                     EGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPD
                     NARR"
     CDS             2102..3202
                     /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     3176..3199
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4013..4036
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4046..4069
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4070..4087
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4114..4132)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4128..4175
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4396..5055)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4970..4989
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5056..5158)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5550..5567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5684..6229)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(5764..5783)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
       61 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
      121 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
      181 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
      241 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
      301 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
      361 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
      421 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
      481 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
      541 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
      601 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
      661 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
      721 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
      781 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
      841 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
      901 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
      961 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     1021 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     1081 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     1141 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     1201 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     1261 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     1321 attaggaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta
     1381 gaaataattt tgtttaactt taataaggag atataccatg agtgcgatta agccagacat
     1441 gaagatcaaa ctccgtatgg aaggcaacgt aaacgggcac cactttgtga tcgacggaga
     1501 tggtacaggc aagccttttg agggaaaaca gagtatggat cttgaagtca aagagggcgg
     1561 acctctgcct tttgcctttg atatcctgac cactgcattc cattacggca acagggtatt
     1621 cgccaaatat ccagacaaca tacaagacta ttttaagcag tcgtttccta aggggtattc
     1681 gtgggaacga agcttgactt tcgaagacgg gggcatttgc aacgccagaa acgacataac
     1741 aatggaaggg gacactttct ataataaagt tcgattttat ggtaccaact ttcccgccaa
     1801 tggtccagtt atgcagaaga agacgctgaa atgggagccc tccactgaga aaatgtatgt
     1861 gcgtgatgga gtgctgacgg gtgatattga gatggctttg ttgcttgaag gaaatgccca
     1921 ttaccgatgt gacttcagaa ctacttacaa agctaaggag aagggtgtca agttaccagg
     1981 cgcccacttt gtggaccact gcattgagat tttaagccat gacaaagatt acaacaaggt
     2041 taagctgtat gagcatgctg ttgctcattc tggattgcct gacaatgcca gacgagggtc
     2101 catgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg
     2161 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca
     2221 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat
     2281 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat
     2341 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta
     2401 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa
     2461 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact
     2521 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc
     2581 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa
     2641 agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat
     2701 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa
     2761 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa
     2821 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt
     2881 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt
     2941 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct
     3001 gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc
     3061 cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt
     3121 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga
     3181 agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa
     3241 caacctcggg attatggctg caagtgagat aatgaacaat ctgcctatgc attccctcga
     3301 ttcttctcta agggatttac ttaacgatga cttattcatt gaaagtgatg aatcgaccaa
     3361 gtcggtaaat gatcagagga gtgaagtatt tcaagaatgt gtgaaccttt tcataaaaag
     3421 agatatcaag gattgcctag aaaaaatgtc tgaggttgga tttattgata ttaccgtttt
     3481 caagtccaac cctatgatat tagatttatt tgtcagcgcc tgtgatatta tgcccagctt
     3541 taccaaatta ggtttaacac tgcagagtga gattttgaat attttcacgt tagatacccc
     3601 tcaatgtatt gagacaagaa aaattattct cggtgacctc agtaaacttt tggtcatcaa
     3661 taaattcttt cgatgctgta tcaaggttat tcagtttaac ttaactgatc acaccgaaca
     3721 agaagaaaaa actctggagc ttgaatccat aatgagtgat ttcattttcg tatacataac
     3781 gaaaatgagg acaactatag atgtagttgg cttacaagaa ttaatcgaga tattcatctt
     3841 tcaggtgaag gtaaaactgc atcataaaaa accctcaccg aatatgtatt gggcgctttg
     3901 caaaacatta cctaagttat ctccaaccct aaagggcctt tacttatcta aagatgtttc
     3961 catagaggat gctattctga actcaataga taataaaata caaaaagata agctggaagt
     4021 cctgttccaa ggtccgggca gcagcgatta taaggatgac gatgacaagc atcaccatca
     4081 tcaccactga ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc
     4141 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca tttgagaagc
     4201 acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg
     4261 ctatttaacg accctgccct gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc
     4321 attcatccgc ttattatcac ttattcaggc gtagcaacca ggcgtttaag ggcaccaata
     4381 actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt
     4441 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg
     4501 catcagcacc ttgtcgcctt gcgtataata tttgcccata gtgaaaacgg gggcgaagaa
     4561 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga
     4621 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca
     4681 cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca
     4741 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc
     4801 ccatatcacc agctcaccgt ctttcattgc catacggaac tccggatgag cattcatcag
     4861 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt
     4921 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg
     4981 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt
     5041 gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     5101 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac
     5161 gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt
     5221 tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg
     5281 tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct ccagtggctt
     5341 ctgtttctat cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt aacggcaaaa
     5401 gcaccgccgg acatcagcgc tagcggagtg tatactggct tactatgttg gcactgatga
     5461 gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag
     5521 aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt
     5581 cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg
     5641 aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc
     5701 cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac
     5761 tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct
     5821 ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca
     5881 ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag
     5941 tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca
     6001 aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg
     6061 cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt
     6121 acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt
     6181 tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct
     6241 tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc
     6301 tgaagtcagc cccatacgat ataagttgt
//
