LOCUS       sequence-325636-        6090 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express recombinant MazF K56A point mutant protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6090)
  AUTHORS   Zhang Z, Chen LQ, Zhao YL, Yang CG, Roundtree IA, Zhang Z, Ren J, 
            Xie W, He C, Luo GZ
  TITLE     Single-base mapping of m(6)A by an antibody-independent method.
  JOURNAL   Sci Adv. 2019 Jul 3;5(7):eaax0250. doi: 10.1126/sciadv.aax0250. 
            eCollection 2019 Jul.
  PUBMED    31281898
REFERENCE   2  (bases 1 to 6090)
  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"; journalName: "Sci Adv."; 
            date: "2019-07-3"; volume: "5(7):eaax0250. doi"; pages: " 
            10.1126/sciadv.aax0250. eCollection 2019 Jul"
COMMENT     Sequence Label: MazF-K56A
FEATURES             Location/Qualifiers
     source          1..6090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..67
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    70..86
                     /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)."
     5'UTR           108..241
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             242..256
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     CDS             257..274
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             275..1570
                     /codon_start=1
                     /product="ribosome-associated chaperone from E. coli 
                     (Hoffmann et al., 2010)"
                     /label=Trigger Factor
                     /translation="MQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFR
                     KGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFT
                     YSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDR
                     VTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYH
                     AENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSA
                     IRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQ
                     AKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMR
                     NVALEEQAVEAVLAKAKVTEKETTFNELMNQQA"
     CDS             1580..1603
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1613..1630
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1637..1648
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     3'UTR           2037..2181
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     primer_bind     complement(2222..2244)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2344..2363
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2380..2835)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2517..2538)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2729..2748
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2935..3039
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3040..3108,3109..3900)
                     /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     complement(3258..3277)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4071..4659
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4560..4579
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4831..5913)
                     /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     5875..5894
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5914..5991)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccgattaatc ataaatatga aaaataattg ttgcatcacc cgccaatgcg tggcttaatg
       61 cacatcaaat tgtgagcgga taacaatttg atgtgctagc gcatatccag tgtagtaagg
      121 caagtccctt caagagttat cgttgatacc cctcgtagtg cacattcctt taacgcttca
      181 aaatctgtaa agcacgccat atcgccgaaa ggcacactta attattaaga ggtaatacac
      241 catgaatcac aaagtgcatc atcatcatca tcacatgcaa gtttcagttg aaaccactca
      301 aggccttggc cgccgtgtaa cgattactat cgctgctgac agcatcgaga ccgctgttaa
      361 aagcgagctg gtcaacgttg cgaaaaaagt acgtattgac ggcttccgca agggcaaagt
      421 gccaatgaat atcgttgctc agcgttatgg cgcgtctgta cgccaggacg ttctgggtga
      481 cctgatgagc cgtaacttca ttgacgccat cattaaagaa aaaatcaatc cggctggcgc
      541 accgacttat gttccgggcg aatacaagct gggtgaagac ttcacttact ctgtagagtt
      601 tgaagtttat ccggaagttg aactgcaagg tctggaagcg atcgaagttg aaaaaccgat
      661 cgttgaagtg accgacgctg acgttgacgg catgctggat actctgcgta aacagcaggc
      721 gacctggaaa gaaaaagacg gcgctgttga agcagaagac cgcgtgacca tcgacttcac
      781 cggttctgta gacggcgaag agttcgaagg cggtaaagcg tctgatttcg tactggcgat
      841 gggccagggt cgtatgatcc cgggctttga agacggtatc aaaggccaca aagctggcga
      901 agagttcacc atcgacgtga ccttcccgga agaataccac gcagaaaacc tgaaaggtaa
      961 agcagcgaaa ttcgctatca acctgaagaa agttgaagag cgtgaactgc cggaactgac
     1021 cgcagagttc atcaaacgtt tcggcgttga agatggttcc gtagaaggtc tgcgcgctga
     1081 agtgcgtaaa aacatggagc gcgagctgaa gagcgccatc cgtaaccgcg ttaagtctca
     1141 ggcgatcgaa ggtctggtaa aagctaacga catcgacgta ccggctgcgc tgatcgacag
     1201 cgaaatcgac gttctgcgtc gccaggctgc acagcgtttc ggtggcaacg aaaaacaagc
     1261 tctggaactg ccgcgcgaac tgttcgaaga acaggctaaa cgccgcgtag ttgttggcct
     1321 gctgctgggc gaagttatcc gcaccaacga gctgaaagct gacgaagagc gcgtgaaagg
     1381 cctgatcgaa gagatggctt ctgcgtacga agatccgaaa gaagttatcg agttctacag
     1441 caaaaacaaa gaactgatgg acaacatgcg caatgttgct ctggaagaac aggctgttga
     1501 agctgtactg gcgaaagcga aagtgactga aaaagaaacc actttcaacg agctgatgaa
     1561 ccagcaggcg tccgcgggtc tggaagttct gttccagggg ccctccgcgg gtctggtgcc
     1621 acgcggtagt ggtggtatcg aaggtaggca tatggtaagc cgatacgtac ccgatatggg
     1681 cgatctgatt tgggttgatt ttgacccgac aaaaggtagc gagcaagctg gacatcgtcc
     1741 agctgttgtc ctgagtcctt tcatgtacaa caacaaaaca ggtatgtgtc tgtgtgttcc
     1801 ttgtacaacg caatcagcag gatatccgtt cgaagttgtt ttatccggtc aggaacgtga
     1861 tggcgtagcg ttagctgatc aggtaaaaag tatcgcctgg cgggcaagag gagcaacgaa
     1921 gaaaggaaca gttgccccag aggaattaca actcattaaa gccaaaatta acgtactgat
     1981 tgggtagctc gagggatccg aattcaagct tgtcgacctg cagtctagat aggtaatctc
     2041 tgcttaaaag cacagaatct aagatccctg ccatttggcg gggatttttt tatttgtttt
     2101 caggaaataa ataatcgatc gcgtaataaa atctattatt atttttgtga agaataaatt
     2161 tgggtgcaat gagaatgcgc aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     2221 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     2281 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     2341 atgcggcatc agagcagatt gtactgagag tgcaccataa aattgtaaac gttaatattt
     2401 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     2461 tcggcaaaat cccttataaa tcaaaagaat agcccgagat agggttgagt gttgttccag
     2521 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     2581 tctatcaggg cgatggccca ctacgtgaac catcacccaa atcaagtttt ttggggtcga
     2641 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2701 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2761 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2821 cgctacaggg cgcgtactat ggttgctttg acgtatgcgg tgtgaaatac cgcacagatg
     2881 cgtaaggaga aaataccgca tcaggcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2941 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3001 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3061 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3121 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3181 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3241 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3301 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3361 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3421 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3481 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3541 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3601 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3661 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3721 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3781 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3841 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3901 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3961 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4021 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4081 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4141 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4201 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4261 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4321 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4381 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4441 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4501 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4561 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4621 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4681 ttacggttcc tggccttttg ctggcctttt gctcacatag tcatgccccg cgcccaccgg
     4741 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4801 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4861 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4921 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4981 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     5041 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     5101 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     5161 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     5221 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     5281 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     5341 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     5401 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5461 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5521 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5581 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5641 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5701 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5761 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5821 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5881 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5941 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     6001 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     6061 tgagcaccgc cgccgcaagg aatggtgtgg
//
