LOCUS       sequence-431864-        6575 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express METTL17-8XHIS in C41(DE3) cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6575)
  AUTHORS   Ast T, Itoh Y, Sadre S, McCoy JG, Namkoong G, Wengrod JC, Chicherin 
            I, Joshi PR, Kamenski P, Suess DLM, Amunts A, Mootha VK
  TITLE     METTL17 is an Fe-S cluster checkpoint for mitochondrial translation.
  JOURNAL   Mol Cell. 2024 Jan 18;84(2):359-374.e8. doi: 
            10.1016/j.molcel.2023.12.016. Epub 2024 Jan 9.
  PUBMED    38199006
REFERENCE   2  (bases 1 to 6575)
  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: 
            "10.1016/j.molcel.2023.12.016"; journalName: "Mol Cell"; date: 
            "2024-01-18- 18"; volume: "84"; issue: "2"; pages: "359-374.e8"
COMMENT     Sequence Label: pET-30a-METTL17-8XHIS
FEATURES             Location/Qualifiers
     source          1..6575
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /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     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3489..3508
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3489..3507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3508..3532
                     /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             3547..3569
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      4751..4760
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4867..4887
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4888..4911
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             4924..4941
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4994..5012)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5008..5055
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5092..5547
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5179..5198)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5389..5410
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5639..6454)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5639..6454)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6362..6381
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6446..6465
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc
     3481 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     3541 aataattttg tttaacttta agaaggagat atacatatgg cagcgctggt gccgggcgtg
     3601 acccaagtgg ataacaaaag cggctttctg cagaaacgcc cgcatcgtca gcatccgggc
     3661 attctgaaac tgcctcatgt gcggctaccg caagcgttag ccaacggagc acagctgctg
     3721 ctgctgggca gcgcgggccc gaccatggaa aaccaagtgc agaccctgac gagctatctg
     3781 tggagccgcc atctgccggt ggaaccggaa gaactgcagc gccgcgcgcg ccatctggaa
     3841 aaaaaatttc tggaaaaccc ggatctgagt cagaccgaag aaaaactgcg cggcgcggtg
     3901 ctgcatgcgc tgcgcaaaac cacctatcat tggcaagaac tgagctatac cgaaggcctg
     3961 agcctggtgt atatggcggc gcgcctggat ggcggctttg cggcggtgag ccgcgcgttt
     4021 catgaaattc gcgcgcgcaa cccggcgttt cagccgcaga ccctgatgga ttttggcagc
     4081 ggcaccggca gcgtgacctg ggcggcgcat agcatttggg gtcagagcct gcgcgaatat
     4141 atgtgcgtgg atcgcagcgc ggcgatgctg gtgctggcgg aaaaactgct gaaaggcggc
     4201 agcgaaagcg gcgaaccgta tattccgggc gtgttttttc gtcagtttct gccggtgagc
     4261 ccgaaagtgc agtttgatgt ggtggtgagc gcgtttagcc tgagcgaact gccgagcaaa
     4321 gcggatcgca ccgaagtggt tcagaccctg tggcgcaaaa ccggccattt tctggtgctg
     4381 gtggaaaacg gcaccaaagc gggccatagc ctgctgatgg atgcgcgcga tctggtgctg
     4441 aaaggcaaag aaaaaagccc gctggatccg cgcccgggct ttgtgtttgc gccgtgtccg
     4501 catgaactgc cgtgcccgca gctgaccaac ctggcgtgca gctttagcca agcgtatcat
     4561 ccgattccgt ttagctggaa caaaaaaccg aaagaagaaa aatttagcat ggtgattctg
     4621 gcgcgcggca gcccggaaga agcgcatcgc tggccgcgca ttacgcagcc ggtgctgaaa
     4681 cgcccgcgcc atgtgcattg ccatctgtgc tgcccggatg gccacatgca gcatgcggtg
     4741 ctgaccgcgc gccgccatgg ccgcgatctg tataggtgtg cgcgcgtgag cagctgggga
     4801 gatctgttgc cggtgctgac cccgagcgct tttccgccga gcaccgcgca agatccgagc
     4861 gaaagcgaaa acctgtattt tcaaggccat catcatcatc atcatcatca ttaataactc
     4921 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     4981 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     5041 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     5101 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5161 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5221 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5281 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     5341 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5401 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     5461 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5521 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     5581 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     5641 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     5701 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     5761 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     5821 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     5881 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     5941 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     6001 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     6061 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     6121 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     6181 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     6241 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     6301 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     6361 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     6421 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     6481 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     6541 gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
