LOCUS       sequence-337861-        6700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the SNAP-tagged MIDAS domain of S. pombe Mdn1 (a.a. 
            4381-4717) without the loop region (a.a. 4458-4496) in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6700)
  AUTHORS   Mickolajczyk KJ, Olinares PDB, Niu Y, Chen N, Warrington SE, Sasaki 
            Y, Walz T, Chait BT, Kapoor TM
  TITLE     Long-range intramolecular allostery and regulation in the 
            dynein-like AAA protein Mdn1.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Aug 4;117(31):18459-18469. doi: 
            10.1073/pnas.2002792117. Epub 2020 Jul 21.
  PUBMED    32694211
REFERENCE   2  (bases 1 to 6700)
  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.2002792117"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2020-08-4- 4"; volume: "117"; issue: "31"; pages: 
            "18459-18469"
COMMENT     Sequence Label: deltaLoop-MIDAS
FEATURES             Location/Qualifiers
     source          1..6700
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     10..29
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        10..28
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    29..53
                     /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             68..90
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             109..126
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             166..186
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             187..732
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             1675..1719
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1757..1775)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1771..1818
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1855..2310
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1942..1961)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2152..2173
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2425..3216,3217..3285))
                     /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     3048..3067
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3286..3378)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3459..4047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3948..3967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4201..4218
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4233..4372
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4298..4317)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4458..4480
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4474..4665)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5237..6319)
                     /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     6281..6300
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6320..6397)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6603..6622
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
       61 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcaccatcat
      121 caccacagcc aggatccgaa ttcggattac gatatcccaa cgaccgaaaa cctgtatttt
      181 cagggcatgg acaaagattg cgaaatgaaa cgtaccaccc tggatagccc gctgggcaaa
      241 ctggaactga gcggctgcga acagggcctg catgaaatta aactgctggg taaaggcacc
      301 agcgcggccg atgcggttga agttccggcc ccggccgccg tgctgggtgg tccggaaccg
      361 ctgatgcagg cgaccgcgtg gctgaacgcg tattttcatc agccggaagc gattgaagaa
      421 tttccggttc cggcgctgca tcatccggtg tttcagcagg agagctttac ccgtcaggtg
      481 ctgtggaaac tgctgaaagt ggttaaattt ggcgaagtga ttagctatca gcagctggcg
      541 gccctggcgg gtaatccggc ggccaccgcc gccgttaaaa ccgcgctgag cggtaacccg
      601 gtgccgattc tgattccgtg ccatcgtgtg gttagctcta gcggtgcggt tggcggttat
      661 gaaggtggtc tggcggtgaa agagtggctg ctggcccatg aaggtcatcg tctgggtaaa
      721 ccgggtctgg gaaagcttgc ggccgctggt gaagataccc ttccaacgga atttgggtct
      781 atcaaccagt ctgagaaggt ttttgaatta tcggaggatg aggatattga agatgaactt
      841 cctgattata atgtgaagat tactaacctt cctgctgcaa tgcccattga tgaagctcgt
      901 gatttatgga ataaacacga ggactctaca aagcaacttt ctattgagtt atgtgaacag
      961 cttcgattga tattagaagc tagcggatct ggatctgtta aacctagtaa aaggacgtat
     1021 caagtgatga tttccattga tgattctaag tcgatgagtg agtcgggcag cacagttcta
     1081 gcgttagaga ctttagcttt ggtaacaaaa gcattatcat tacttgaagt tggacaaatt
     1141 gcagtgatga aatttgggga gcaacctgaa ttgttgcatc ctttcgacaa acagttttca
     1201 tctgaaagtg gtgtacaaat gttttcgcat tttacatttg agcaaagtaa taccaatgtt
     1261 ttggctctcg ccgatgcttc tatgaaatgc tttaactatg ccaatacggc ttcccatcat
     1321 agatctaact cggacataag gcagttagaa atcataattt cagacggtat ttgtgaagat
     1381 catgatagca ttcggaaact gctaaggcgt gctcaagaag aaaaagtgat gattgtgttt
     1441 gttattcttg acaatgttaa cacccagaaa aagtcgtcca tccttgatat taaaaaggtt
     1501 tactacgaca ctaaagaaga tggtactatg gatttaaaaa ttcagccata tatagatgag
     1561 tttgcttttg attactattt ggtggttcgt aacatcgaag aattgccgca acttttgtct
     1621 tctgcacttc gtcaatggtt tcagcagatg agtaatactt aactcgagtc tggtaaagaa
     1681 accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta
     1741 acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1801 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     1861 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1921 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1981 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2041 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2101 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2161 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2221 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2281 attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga gattatcaaa
     2341 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     2401 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     2461 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2521 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     2581 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     2641 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     2701 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     2761 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2821 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2881 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     2941 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3001 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     3061 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     3121 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     3181 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3241 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3301 atcatgattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     3361 tttagaaaaa taaacaaata ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3421 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3481 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3541 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3601 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3661 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3721 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3781 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3841 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3901 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3961 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4021 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4081 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4141 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4201 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     4261 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     4321 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     4381 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4441 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4501 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     4561 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     4621 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     4681 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     4741 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     4801 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     4861 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     4921 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     4981 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     5041 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     5101 caacgacagg agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg
     5161 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5221 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5281 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5341 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5401 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5461 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     5521 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5581 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5641 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5701 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5761 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5821 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5881 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5941 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6001 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     6061 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     6121 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6181 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     6241 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     6301 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     6361 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     6421 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     6481 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     6541 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6601 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     6661 acgatgcgtc cggcgtagag gatcgagatc gatctcgatc
//
