LOCUS       sequence-389368-        7086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis Plasmid: pTet, danQ926, dam, seqA, emrR, ugi and cda1, 
            tetR, CloDF13, KanR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7086)
  AUTHORS   Al'Abri IS, Haller DJ, Li Z, Crook N
  TITLE     Inducible directed evolution of complex phenotypes in bacteria.
  JOURNAL   Nucleic Acids Res. 2022 Jun 10;50(10):e58. doi: 10.1093/nar/gkac094.
  PUBMED    35150576
REFERENCE   2  (bases 1 to 7086)
  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: "Nucleic 
            Acids Res."; date: "2022-06-10"; volume: "50(10):e58. doi"; pages: "
            10.1093/nar/gkac094"
COMMENT     Sequence Label: ISA431: aTc-MP6
FEATURES             Location/Qualifiers
     source          1..7086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(501..1316)
                     /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(501..1316)
                     /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     1224..1243
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1308..1327
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1511..1529
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1536..1554
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             4368..4622
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4652..5278
                     /codon_start=1
                     /product="cytidine deaminase from the sea lamprey 
                     Petromyzon marinus (Rogozin et al., 2007)"
                     /label=PmCDA1
                     /translation="MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGER
                     RACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKI
                     LEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIF
                     IQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV"
     terminator      complement(5305..5336)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             5484..6107
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(5557..5576)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(6297..6316)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      complement(join(6723..7086,1..375))
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
ORIGIN
        1 gtcgttcgct ccaagctggg ctgtaagcaa gaactccccg ttcagcccga ctgctgcgcc
       61 ttatccggta actgttcact tgagtccaac ccggaaaagc acggtaaaac gccactggca
      121 gcagccattg gtaactggga gttcgcagag gatttgttta gctaaacacg cggttgctct
      181 tgaagtgtgc gccaaagtcc ggctacactg gaaggacaga tttggttgct gtgctctgcg
      241 aaagccagtt accacggtta agcagttccc caactgactt aaccttcgat taaaccacct
      301 ccccaggtgg ttttttcgtt tacagggcaa aagattacgc gcagaaaaaa aggatctcaa
      361 gaagatcctt tgatcttttc tactgaaccg ctctagattt cagtgcaatt tatctcttca
      421 aatgtagcac ctgaagtcag cccagtggaa cgaaaaatca atctaaagta tatatgagta
      481 aacttggtct gacagttacc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
      541 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
      601 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
      661 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
      721 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg catttctttc
      781 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
      841 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
      901 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
      961 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     1021 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     1081 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     1141 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     1201 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     1261 atgttggaat ttaatcgcgg cctggagcaa gacgtttccc gttgaatatg gctcataaca
     1321 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta
     1381 tcttgtgcaa tgtacatcag agattttgag acacaaccaa ttattgaagg cctccctaac
     1441 ggggggcctt tttttgtttc tggtctcccg cttaacgatc gttggctgtt ttcagcagga
     1501 cgcactgacc tccctatcag tgatagagat tgacatccct atcagtgata gagatactga
     1561 acccgttttt ttggacgcgt acaactcaag tctgacataa atgaccgcta tgagcactgc
     1621 aattacacgc cagatcgttc tcgctaccgc aaccaccggt atgaaccaga ttggtgcgca
     1681 ctatgaaggc cacaagatca ttgagattgg tgccgttgaa gtggtgaacc gtcgcctgac
     1741 gggcaataac ttccatgttt atctcaaacc cgatcggctg gtggatccgg aagcctttgg
     1801 cgtacatggt attgccgatg aatttttgct cgataagccc acgtttgccg aagtagccga
     1861 tgagttcatg gactatattc gcggcgcgga gttggtgatc cataacgcag cgttcgatat
     1921 cggctttatg gactacgagt tttcgttgct taagcgcgat attccgaaga ccaatacttt
     1981 ctgtaaggtc accgatagcc ttgcggtggc gaggaaaatg tttcccggta agcgcaacag
     2041 cctcgatgcg ttatgtgctc gctacgaaat agataacagt aaacgaacgc tgcacggggc
     2101 attactcgat gcccagatcc ttgcggaagt ttatctggcg atgaccggtg gtcaaacgtc
     2161 gatggctttt gcgatggaag gagagacaca acagcaacaa ggtgaagcaa caattcagcg
     2221 cattgtacgt caggcaagta agttacgcgt tgtttttgcg acagatgaag agattgcagc
     2281 tcatgaagcc cgtctcgatc tggtgcagaa gaaaggcgga agttgcctct ggcgagcata
     2341 atttaatatc agtaaaccgg acataaccca tgaagaaaaa tcgcgctttt ttgaagtggg
     2401 cagggggcaa gtatcccctg cttgatgata ttaaacggca tttgcccaag ggcgaatgtc
     2461 tggttgagcc ttttgtaggt gccgggtcgg tgtttctcaa caccgacttt tctcgttata
     2521 tccttgccga tatcaatagc gacctgatca gtctctataa cattgtgaag atgcgtactg
     2581 atgagtacgt acaggccgca cgcgagctgt ttgttcccga aacaaattgc gccgaggttt
     2641 actatcagtt ccgcgaagag ttcaacaaaa gccaggatcc gttccgtcgg gtggtactgt
     2701 ttttatattt gaaccgctac ggttacaacg gcctgtgtcg ttacaatctg cgcggtgagt
     2761 ttaacgtgcc gttcggccgc tacaaaaaac cctatttccc ggaagcagag ttgtatcact
     2821 tcgctgaaaa agcgcagaat gcctttttct attgtgagtc ttacgccgat agcatggcgc
     2881 gcgcagatga tgcatccgtc gtctattgcg atccgcctta tgcaccgctg tctgcgaccg
     2941 ccaactttac ggcgtatcac acaaacagtt ttacgcttga acaacaagcg catctggcgg
     3001 agatcgccga aggtctggtt gagcgccata ttccagtgct gatctccaat cacgatacga
     3061 tgttaacgcg tgagtggtat cagcgcgcaa aattgcatgt cgtcaaagtt cgacgcagta
     3121 taagcagcaa cggcggcaca cgtaaaaagg tggacgaact gctggctttg tacaaaccag
     3181 gagtcgtttc acccgcgaaa aaataattca gctaagacac tgcactggat taagatgaaa
     3241 acgattgaag ttgatgatga actctacagc tatattgcca gccacactaa gcatatcggc
     3301 gagagcgcat ccgacatttt acggcgtatg ttgaaatttt ccgccgcatc acagcctgct
     3361 gctccggtga cgaaagaggt tcgcgttgcg tcacctgcta tcgtcgaagc gaagccggtc
     3421 aaaacgatta aagacaaggt tcgcgcaatg cgtgaacttc tgctttcgga tgaatacgca
     3481 gagcaaaagc gagcggtcaa tcgctttatg ctgctgttgt ctacactata ttctcttgac
     3541 gcccaggcgt ttgccgaagc aacggaatcg ttgcacggtc gtacacgcgt ttactttgcg
     3601 gcagatgaac aaacgctgct gaaaaatggt aatcagacca agccgaaaca tgtgccaggc
     3661 acgccgtatt gggtgatcac caacaccaac accggccgta aatgcagcat gatcgaacac
     3721 atcatgcagt cgatgcaatt cccggcggaa ttgattgaga aggtttgcgg aactatctaa
     3781 cggctgaaat taatgaggtc atacccaaat ggatagttcg tttacgccca ttgaacaaat
     3841 gctaaaattt cgcgccagcc gccacgaaga ttttccttat caggagatcc ttctgactcg
     3901 tctttgcatg cacatgcaaa gcaagctgct ggagaaccgc aataaaatgc tgaaggctca
     3961 gggaattaac gagacgttgt ttatggcgtt gattacgctg gagtctcagg aaaaccacag
     4021 tattcagcct tctgaattaa gttgtgctct tggatcatcc cgtaccaacg cgacgcgtat
     4081 tgccgatgaa ctggaaaaac gcggttggat cgaacgtcgt gaaagcgata acgatcgccg
     4141 ctgcctgcat ctgcaattaa cggaaaaagg tcacgagttt ttgcgcgagg ttttaccacc
     4201 gcagcataac tgcctgcatc aactctggtc cgcgctcagc acaacagaaa aagatcagct
     4261 cgagcaaatc acccgcaaat tgctctcccg tctcgaccag atggaacaag acggtgtggt
     4321 tctcgaagcg atgagctaat aatacaaaaa ttaggaggaa tttcaacatg acaaatttat
     4381 ctgacatcat tgaaaaagaa acaggaaaac aactagtgat tcaagaatca attctaatgt
     4441 taccagaaga agtagaggaa gtaattggga ataaaccaga aagtgatatt ttagttcata
     4501 ctgcttatga tgaaagtaca gatgaaaatg taatgctatt aacttcagat gctccagaat
     4561 ataaaccttg ggctttagta attcaagaca gtaatggaga aaataaaatt aaaatgttat
     4621 aagtcgagat taagtaaacc ggaatctgaa gatgaccgac gcggaatacg ttcgtatcca
     4681 cgaaaaactg gacatctaca ccttcaaaaa acagttcttc aacaacaaaa aatctgtttc
     4741 tcaccgttgc tacgttctgt tcgaactgaa acgtcgtggt gaacgtcgtg cgtgcttctg
     4801 gggttacgcg gttaacaaac cgcagtctgg taccgaacgt ggtatccacg cggaaatctt
     4861 ctctatccgt aaagttgaag aatacctgcg tgacaacccg ggtcagttca ccatcaactg
     4921 gtactcttct tggtctccgt gcgcggactg cgcggaaaaa atcctggaat ggtacaacca
     4981 ggaactgcgt ggtaacggtc acaccctgaa aatctgggcg tgcaaactgt actacgaaaa
     5041 aaacgcgcgt aaccagatcg gtctgtggaa cctgcgtgac aacggtgttg gtctgaacgt
     5101 tatggtttct gaacactacc agtgctgccg taaaatcttc atccagtctt ctcacaacca
     5161 gctgaacgaa aaccgttggc tggaaaaaac cctgaaacgt gcggaaaaac gtcgttctga
     5221 actgtctatc atgatccagg ttaaaatcct gcacaccacc aaatctccgg cggtttaaac
     5281 ttaattaacg gcactcctca gccaagtcaa aagcctccgg tcggaggctt ttgactctcg
     5341 gtaccaaatt ccagaaaaga ggcctcccga aaggggggcc ttttttcgtt ttggtccaat
     5401 ggcggcgcgc catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     5461 agagtcaatt cagggtggtg aatatgtcca gattagataa aagtaaagtg attaacagcg
     5521 cattagagct gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga
     5581 agctaggtgt agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg
     5641 acgccttagc cattgagatg ttagataggc accatactca cttttgccct ttagaagggg
     5701 aaagctggca agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc
     5761 atcgcgatgg agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc
     5821 tcgaaaatca attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg
     5881 cactcagcgc tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag
     5941 tcgctaaaga agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag
     6001 ctatcgaatt atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga
     6061 tcatatgcgg attagaaaaa caacttaaat gtgaaagtgg gtcctgataa ggatcctaat
     6121 tggtaacgaa tcagaattga cggctcgagg gagtagcata gggtttgcag aatccctgct
     6181 tcgtccattt gacaggcaca ttatgcatcg atgataagct gtcaaacatg agcagatcct
     6241 ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta
     6301 tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcaaata
     6361 ttttatctga ggtgcttcct cgctcactga ctcgctgcac gaggcagacc tcagcgctag
     6421 cggagtgtat actggcttac tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg
     6481 tggcaggaga aaaaaggctg caccggtgcg tcagcagaat atgtgataca ggatatattc
     6541 cgcttcctca ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc
     6601 ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag
     6661 tacgtgccaa gccaaatagg ccgtcactcg gtcgctacgc tccgggcgtg agactgcggc
     6721 gggcgctgcg gacacataca aagttaccca cagattccgt ggataagcag gggactaaca
     6781 tgtgaggcaa aacagcaggg ccgcgccggt ggcgtttttc cataggctcc gccctcctgc
     6841 cagagttcac ataaacagac gcttttccgg tgcatctgtg ggagccgtga ggctcaacca
     6901 tgaatctgac agtacgggcg aaacccgaca ggacttaaag atccccaccg tttccggcgg
     6961 gtcgctccct cttgcgctct cctgttccga ccctgccgtt taccggatac ctgttccgcc
     7021 tttctccctt acgggaagtg tggcgctttc tcatagctca cacactggta tctcggctcg
     7081 gtgtag
//
