LOCUS       sequence-226772-        6331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector with mazEF toxin antitoxin system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6331)
  AUTHORS   Diebold-Durand ML, Burmann F, Gruber S
  TITLE     High-Throughput Allelic Replacement Screening in Bacillus subtilis.
  JOURNAL   Methods Mol Biol. 2019;2004:49-61. doi: 10.1007/978-1-4939-9520-2_5.
  PUBMED    31147909
REFERENCE   2  (bases 1 to 6331)
  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: "Methods Mol
            Biol."; date: "2019"; volume: "2004:49-61. doi"; pages: " 
            10.1007/978-1-4939-9520-2_5"
COMMENT     Sequence Label: pET-Gate2 mazEF
FEATURES             Location/Qualifiers
     source          1..6331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     primer_bind     complement(1266..1284)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1280..1327
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1364..1819
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1451..1470)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1661..1682
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1911..2726)
                     /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(1911..2726)
                     /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     2634..2653
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2718..2737
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2848..3436
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3337..3356
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3590..3607
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3622..3761
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3687..3706)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3847..3869
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3863..4054)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4863..5945)
                     /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     5907..5926
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5946..6023)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6229..6248
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gtctcgagag
       61 aaacttctct caattaagaa aaaggcccca acatcttggg gcctttttct tttttatcta
      121 acaatgacta cccaatcagt acgttaattt tggctttaat gagttgtaat tcctctgggg
      181 caactgttcc tttcttcgtt gctcctcttg cccgccaggc gatacttttt acctgatcag
      241 ctaacgctac gccatcacgt tcctgaccgg ataaaacaac ttcgaacgga tatccttttg
      301 attgcgttgt acaaggaaca cacagacaca tacctgtttt gttgttgtac atgaaaggac
      361 tcaggacaac agctggacga tgtccagctt gctcgctacc ttttgtcggg tcaaaatcaa
      421 cccaaatcag atcgcccata tcgggtacgt atcggcttac catcaaaatc gtctccctcc
      481 gtttgaatat ttgattgatc gtaaccagat gaagcactct ttccactatc cctacagtgt
      541 tatggcttga acaatcacga aacaataatt ggtacgtacg atctttcagc cgactcaaac
      601 atcaaatctt acaaatgtag tctttgaaag tattacatat gtaagattta aatgcaaccg
      661 ttttttgcgg ccgccgagag agacccagaa acttctctca attaagaaaa aggccccaac
      721 atcttggggc ctttttcttt tttatctatt accagacttc cttatctttc ggctctcccc
      781 agtcgatatt ctcgtggagg ttttccggcg tgatgtcgtt gaccagttca gcaagcgtaa
      841 atacgggctc tttacgcact ggctcaataa ttaatttgcc atccaccagg tcaatcttca
      901 cttcatcatc aatattcaga ttgagcgcct gcattaacgt agccgggatc cgcaccgccg
      961 gtgaatttcc ccaacgcttt acgctactgt ggatcatcaa aatcgtctcc ctccgtttga
     1021 atatttgatt gatcgtaacc agatgaagca ctctttccac tatccctaca gtgttatggc
     1081 ttgaacaatc acgaaacaat aattggtacg tacgatcttt cagccgactc aaacatcaaa
     1141 tcttacaaat gtagtctttg aaagtattac atatgtaaga tttaaatgca accgtttttt
     1201 ggtctcacca tggtgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     1261 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1321 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     1381 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1441 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1501 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1561 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1621 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1681 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1741 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     1801 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1861 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     1921 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     1981 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     2041 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     2101 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     2161 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     2221 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     2281 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     2341 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     2401 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     2461 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     2521 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     2581 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     2641 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     2701 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     2761 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2821 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2881 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2941 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3001 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3061 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3121 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3181 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3241 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3301 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3361 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3421 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3481 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3541 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3601 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3661 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3721 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3781 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3841 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3901 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3961 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     4021 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4081 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4141 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4201 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4261 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4321 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4381 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4441 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4501 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4561 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4621 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4681 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4741 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4801 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4861 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4921 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4981 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5041 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5101 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5161 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5221 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5281 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5341 tgccagccag ccagacgcag acgcgccgag acagaactta atggtcccgc taacagcgcg
     5401 atttgctgat gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5461 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5521 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5581 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5641 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5701 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5761 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5821 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5881 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5941 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     6001 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6061 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     6121 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     6181 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     6241 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     6301 gtagaggatc gagatctcga tcccgcgaaa t
//
