LOCUS       sequence-445265-        7260 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of ECOR31 Hachiman mutant (HamB D431A) under the pTet 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7260)
  AUTHORS   Tuck OT, Adler BA, Armbruster EG, Lahiri A, Hu JJ, Zhou J, Pogliano 
            J, Doudna JA
  TITLE     Hachiman is a genome integrity sensor.
  JOURNAL   bioRxiv [Preprint]. 2024 Feb 29:2024.02.29.582594. doi: 
            10.1101/2024.02.29.582594.
  PUBMED    38464307
REFERENCE   2  (bases 1 to 7260)
  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: "bioRxiv 
            [Preprint]. 2024 Feb 29:2024.02.29.582594. doi: 
            10.1101/2024.02.29.582594."
COMMENT     Sequence Label: pBA1467_pTet-ECOR31Ham_hamB_walkerB_Mut
FEATURES             Location/Qualifiers
     source          1..7260
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             133..162
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             178..195
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(251..268)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      421..467
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(499..1125)
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     1030..1049
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        1141..1196
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1177..1195
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      complement(5544..5578)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(5649..6308)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6223..6242
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(6547..6564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6681..7226)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(6761..6780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
       61 agttgtcacc tgcccgaacc agagacttat cctgaactgg ccgataattg cagacgaacg
      121 atgcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat
      181 catcatcatc atcattgagt ttaaacgctc tccagcttgg gtgttttggc ggatgagaga
      241 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt
      301 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac
      361 gccgtagcgc cgatggtagt gtggggtctg cccatgcgag agtaggcaac tgccaggcat
      421 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
      481 gtgaacttca gcgacgtctt aagacccact ttcacattta agttgttttt ctaatccgca
      541 tatgatcaat tcaaggccga ataagaaggc tggctctgca ccttggtgat caaataattc
      601 gatagcttgt cgtaataatg gcggcatact atcagtagta ggtgtttccc tttcttcttt
      661 agcgacttga tgctcttgat cttccaatac gcaacctaaa gtaaaatgcc ccacagcgct
      721 gagtgcatat aatgcattct ctagtgaaaa accttgttgg cataaaaagg ctaattgatt
      781 ttcgagagtt tcatactgtt tttctgtagg ccgtgtacct aaatgtactt ttgctccatc
      841 gcgatgactt agtaaagcac atctaaaact tttagcgtta ttacgtaaaa aatcttgcca
      901 gctttcccct tctaaagggc aaaagtgagt atggtgccta tctaacatct caatggctaa
      961 ggcgtcgagc aaagcccgct tattttttac atgccaatac aatgtaggct gctctacacc
     1021 tagcttctgg gcgagtttac gggttgttaa accttcgatt ccgacctcat taagcagctc
     1081 taatgcgctg ttaatcactt tacttttatc taatctagac atcattaatt cctaattttt
     1141 gttgacactc tatcgttgat atagttattt taccactccc tatcagtgat agagaaaaaa
     1201 ttcaaaagat cttttaagaa ggagatatac tgatggcaaa tttcgaggac tggtgtgatt
     1261 cgacagagcg taatatcagc gaccattatt tgcagtcaat aacggccagg gatgccgagt
     1321 gtatgttcgg cgttcaggtt atggccgcgc taatacctga acactatgcg tcaccaagaa
     1381 atatcgctaa cgcatttgaa gctctaggta aaccggggct ggcggcatac attgctggaa
     1441 aattgcctga aacgaaacag attcgttccg gtgatttggg tgagatattt gcaactgaat
     1501 ggataaatgc acgtagcaat ggctataaaa cgcctataaa aagattgcgc tggaaagatc
     1561 atcgcaatat gtcgatgcgt ggtgaggacg tcattggtat ttacatagat cagtcttctc
     1621 aacaactttt tttccttaaa accgaagcga aaagtcgcgc caaaatgacg ggagaggtgg
     1681 tttctgaagc ccgcgataac ttgaataaag agcaagggct gccatcttca catgccctaa
     1741 tgttcatagc agatcgcctg aatgaacagg gtgaggagct tttggctaaa gcgatcttaa
     1801 atgcaacgct tcggcaagga attgttccag gatgcgtccg gcatcttatc tttctattat
     1861 cgggcaactc ttcagaaact atgctgacca cttcaattga aaagtataca ggtcaaaata
     1921 atcagtgggg tgtttgtcta agaattgctc gccatggtga gtttattgcc gcaacttttg
     1981 aaaaggtgat tagcgatgcc agcaacagct gatgaaatta tcgaagctat taaggaagct
     2041 tcagctgtag gttttcgcgg gcgcctgatt gcacgcgggc aagcccggtc ggtgatctgg
     2101 cgagacggtg acttgccgcc ggatgcaccg gagtttagcg cactgctcag tcaggacctt
     2161 caaggctatg cttatgccct aatcgatctc ggccttcgtc ttcgagaact taatggagac
     2221 gacgcctatg cgcgaattgc ttttgaacag gccggaaccg cactcgagtc ggctattgct
     2281 aaaggaaaac gcgacagccg ggatacagat tttcacttcg taatggctgc tgcaagctat
     2341 caccttgcgc atctttcagc acgagcctat tcgttactcg caatggttgg tcaggatgat
     2401 aatttttcac caatagagag ggctcttaca caactgatcc ggcgtgattt gcgtactctg
     2461 cgcgataatg cgttgggctt tcggctcaga ggtgatggca gtgacgtgaa aattactgaa
     2521 atcctccagg ctcgcctaaa cttgccgcaa gacgagaacg gtgacagtga gtcagaggag
     2581 gacatccttt ttgatgggct tgatttggca ctgactgacg cctatatgtc ggccatttct
     2641 ctctaccttc tcgctgtaga aaggggagaa tcacgtttgc tttccagggc tattgaaaaa
     2701 ttaagaatta gtctaagtat atgtgctcag tttaatatgc taccccagtg gtggctaaac
     2761 tttattacca ttcatctgtt atctgacctt tggtcagata cattccatga aagattgcca
     2821 ctggttccag ttggtggtga cgctgctgaa tggcccgcgc ttcgggaact gtttattgca
     2881 ctgcttcaaa gacgacctcg agccgaaatt gatctctggc catcgcagag agaggccgcc
     2941 ggacgctctg ttaatgacaa tgacgatttg gttgtttcgt tgccaaccag cgctggaaaa
     3001 actcgtattg ctgaattgtg tattcttcgc tgtctggcgg gtggcaagcg tgttgttttc
     3061 atcaccccgc ttcgtgcgct ttctgcacaa acagaagcca ctctctccag aacatttggg
     3121 ccgcttggga aaacaatatc aatgctgtat ggcagcattg gcgtaagtgg tatggatgag
     3181 gatgcaattc gccagcgtga catcgtcgtc gccacgcctg aaaaacttga ctttgctctc
     3241 cggaatgacc cgtcaattat taatgatgta ggacttttta tttttgcgga ggggcacatg
     3301 attggggccg atgaacgaga ggttcggtat gaagttcaaa ttcaacggct gttgcgcaga
     3361 caggatgccg atacgcgcag aattgtctgc ctctcggcaa ttctacctga tggtgagcaa
     3421 ctcgatgatt ttgccggttg gctccgtcgt gacaagcccg gaggaccaat caaaaataat
     3481 tggcgaccga cccgcttgca gtttggcgag gtcatatggt ccgctccagc aggtcggctt
     3541 aatctcagcg tcggttatga ggcggcttgg gtatcgcgtt ttatcgtcag cagacaaccg
     3601 ccaaaggtta aattacctaa taaaaagcag cgaacaaaaa tgtttccctc tgataacaag
     3661 gagctctgct tagcgacagc ctggcgtcta attgaagatg gacagaccgt acttatctac
     3721 tgcccacttc gccggagcgt cgaaccgttt gccgagacaa ttgtcgatct gcatcagcgc
     3781 ggactactgc catctttatt tgatgctgcc ccggacattc tggatacggc tatctcgctg
     3841 ggagaagaat ggttgggggc acacagcccg atacttgcct gcctacggtt gggcgtggct
     3901 cttcatcatg gcgcattgcc tacggcttac cgcaaagaaa tcgaacgttt gctgcgagat
     3961 ggggtactta aagtgacgat ctcatcaccg acgctagcac aaggtcttaa tctttcggca
     4021 acagctatcg tcatgcattc tcttcatcgg aatcgcgagc ttatcaaagt gtccgagttc
     4081 agaaacgtca taggcagagc agggcgcgca tatgtagatg ttgaagggct ggtgatttat
     4141 ccaatattcg ataaagtaaa taaacgccag actaactggc ataccctcac atctgacacg
     4201 ggggcccgtg agatggagag cggactaata cagcttgtct gtgtgttatt gattcgcatg
     4261 catacacggc ttggtgggga tttaaaagca ctcactgaat atgtgaccaa caacgccgta
     4321 gcgtgggaat ttccagaaat tatgaccgaa tctccgcaag aaagagatat tgcgcaggcc
     4381 atctgggaaa aacaactttc aacgcttgat actgctattc tgagccttct tggtgagaat
     4441 gatatcccgg atgaccagat tgaaactgct cttgatgaca ttcttcagtc ttcactttgg
     4501 caacgcagtt tgcagcgtta tagggatgag aatgaacgga ttttgctgaa atcgggactc
     4561 ctgtcccgca gccgctacat ctggcagcgc tcaacggcag cagggcgccg ggggtatttc
     4621 ctgtcggggg taggacttac aaccgggcta cgacttgatg ctattgccgc taaggccaac
     4681 caactattga ttgatgcgaa tgcggctata atgggcggtg atgccgaaga ggcaattgct
     4741 gctatcacgg cgctcgctga agaggtcttt accttctatc cgtttatacc cgatccattg
     4801 cctggcgact ggagaggtat tttacgttcc tggctattgg gtgaacccat gactaacgtc
     4861 gcgaatacgc aggcgtctga aacgcttcaa ttcgtcgaga atgggctggt gtatcgactt
     4921 ccgtgggcaa tggaggctat tagggtacgt gcaacagcta atggagacct cataggtgat
     4981 acagatacca cgctagacga ttatgagtta ggttttgcgg ttgctgctgt tgaaacaggt
     5041 accttgagcc gttcgtcttc actgcttatt caggcgggtt ttagctctag gcttgcagcc
     5101 ataaaagtgg ttacagacac tactgccgat tttcaatccg gccaggagct tagaaggtgg
     5161 ctgaactctg aagaagtgat aagccacacg gataatcatg actggcctac acctgaaacc
     5221 agagtgatgt ggctggaatt cttgggtagt ctcagcccca aaggcagcca ggtttggagt
     5281 cgtcatcgtt ataacggaat ggtggactgg agagatactc cggcagtgat cggcacacca
     5341 ttgcagcttt acactgttga tggtattcat cacgtactgg ctgatgatgg cactcctctg
     5401 ggaagtataa atggcagaat taacacaaac agaagaggtc ttcttagagt tgaggtggat
     5461 gatgagaatg gtcgtgccat gttcgattat ttaggaccgg acgactttat ttctacctga
     5521 aggaggactt tggcaggtga ccaaaaaaaa acgcccggcg gcaaccgagc gttctgaaca
     5581 aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca agcgagctcg
     5641 atatcaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt
     5701 ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc
     5761 accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc
     5821 atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgaaacgaaa
     5881 aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca
     5941 tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat
     6001 gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc
     6061 accagctcac cgtctttcat tgccatacga aattccggat gagcattcat caggcgggca
     6121 agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag
     6181 gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc
     6241 tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt
     6301 ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt
     6361 agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgcccgat caagcgctag
     6421 cggagtgtat actggcttac tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg
     6481 tggcaggaga aaaaaggctg caccggtgcg tcagcagaat atgtgataca ggatatattc
     6541 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc
     6601 ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag
     6661 agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca tcacgaaatc
     6721 tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6781 cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg
     6841 ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg
     6901 ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg
     6961 taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac
     7021 tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa
     7081 ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag
     7141 ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga
     7201 ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata aaatatttct
//
