LOCUS       sequence-337859-        6799 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the SNAP-tagged MIDAS domain of S. pombe Mdn1 (a.a. 
            4381-4717) in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6799)
  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 6799)
  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: SNAP-MIDAS
FEATURES             Location/Qualifiers
     source          1..6799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    5..29
                     /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             44..66
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             85..102
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             142..162
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             163..708
                     /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             1750..1794
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1832..1850)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1846..1893
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1930..2385
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2017..2036)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2227..2248
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2500..3291,3292..3360))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRSIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3123..3142
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3361..3453)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3534..4122
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4023..4042
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4276..4293
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4308..4447
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4373..4392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4533..4555
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4549..4740)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5312..6394)
                     /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     6356..6375
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6395..6472)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6678..6697
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6785..6799,1..5)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6785..6799,1..4)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
       61 aggagatata ccatgggcag cagccatcac catcatcacc acagccagga tccgaattcg
      121 gattacgata tcccaacgac cgaaaacctg tattttcagg gcatggacaa agattgcgaa
      181 atgaaacgta ccaccctgga tagcccgctg ggcaaactgg aactgagcgg ctgcgaacag
      241 ggcctgcatg aaattaaact gctgggtaaa ggcaccagcg cggccgatgc ggttgaagtt
      301 ccggccccgg ccgccgtgct gggtggtccg gaaccgctga tgcaggcgac cgcgtggctg
      361 aacgcgtatt ttcatcagcc ggaagcgatt gaagaatttc cggttccggc gctgcatcat
      421 ccggtgtttc agcaggagag ctttacccgt caggtgctgt ggaaactgct gaaagtggtt
      481 aaatttggcg aagtgattag ctatcagcag ctggcggccc tggcgggtaa tccggcggcc
      541 accgccgccg ttaaaaccgc gctgagcggt aacccggtgc cgattctgat tccgtgccat
      601 cgtgtggtta gctctagcgg tgcggttggc ggttatgaag gtggtctggc ggtgaaagag
      661 tggctgctgg cccatgaagg tcatcgtctg ggtaaaccgg gtctgggaaa gcttgcggcc
      721 gctggtgaag atacccttcc aacggaattt gggtctatca accagtctga gaaggttttt
      781 gaattatcgg aggatgagga tattgaagat gaacttcctg attataatgt gaagattact
      841 aaccttcctg ctgcaatgcc cattgatgaa gctcgtgatt tatggaataa acacgaggac
      901 tctacaaagc aactttctat tgagttatgt gaacagcttc gattgatatt agaaccaacc
      961 ttggccacta agatgcaagg agactttcgt acgggtaaac ggttgaatat gaaacgaatc
     1021 atcccatata ttgcttctca gttcaagaaa gacaagatat ggatgcgtcg tgttaaacct
     1081 agtaaaagga cgtatcaagt gatgatttcc attgatgatt ctaagtcgat gagtgagtcg
     1141 ggcagcacag ttctagcgtt agagacttta gctttggtaa caaaagcatt atcattactt
     1201 gaagttggac aaattgcagt gatgaaattt ggggagcaac ctgaattgtt gcatcctttc
     1261 gacaaacagt tttcatctga aagtggtgta caaatgtttt cgcattttac atttgagcaa
     1321 agtaatacca atgttttggc tctcgccgat gcttctatga aatgctttaa ctatgccaat
     1381 acggcttccc atcatagatc taactcggac ataaggcagt tagaaatcat aatttcagac
     1441 ggtatttgtg aagatcatga tagcattcgg aaactgctaa ggcgtgctca agaagaaaaa
     1501 gtgatgattg tgtttgttat tcttgacaat gttaacaccc agaaaaagtc gtccatcctt
     1561 gatattaaaa aggtttacta cgacactaaa gaagatggta ctatggattt aaaaattcag
     1621 ccatatatag atgagtttgc ttttgattac tatttggtgg ttcgtaacat cgaagaattg
     1681 ccgcaacttt tgtcttctgc acttcgtcaa tggtttcagc agatgagtaa tacttaactc
     1741 gagtctggta aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact
     1801 agcgcagctt aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt
     1861 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1921 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1981 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2041 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2101 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2161 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2221 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2281 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2341 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttctggc ggcacgatgg
     2401 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2461 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2521 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2581 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgatactgcg
     2641 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2701 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     2761 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     2821 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     2881 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     2941 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3001 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3061 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3121 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3181 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3241 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3301 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3361 actcttcctt tttcaatcat gattgaagca tttatcaggg ttattgtctc atgagcggat
     3421 acatatttga atgtatttag aaaaataaac aaataggtca tgaccaaaat cccttaacgt
     3481 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3541 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3601 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3661 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3721 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3781 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3841 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3901 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3961 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4021 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4081 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4141 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4201 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4261 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4321 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4381 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4441 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4501 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4561 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4621 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4681 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4741 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4801 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4861 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4921 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4981 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5041 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5101 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5161 gcctagccgg gtcctcaacg acaggagcac gatcatgcta gtcatgcccc gcgcccaccg
     5221 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     5281 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5341 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5401 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     5461 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     5521 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     5581 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     5641 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     5701 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5761 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5821 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5881 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5941 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     6001 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     6061 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     6121 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     6181 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     6241 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     6301 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     6361 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     6421 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     6481 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     6541 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     6601 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     6661 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     6721 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
     6781 aaattaatac gactcacta
//
