LOCUS       sequence-444938-        7261 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of ECOR31 Hachiman mutant (HamA E138A, K140A) under the 
            pTet promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7261)
  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 7261)
  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: pBA1469_pTet-ECOR31Ham_hamA_E138A_K140A_Mut
FEATURES             Location/Qualifiers
     source          1..7261
                     /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(5545..5579)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(5650..6309)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6224..6243
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(6548..6565)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6682..7227)
                     /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(6762..6781)
                     /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 agagaaaaga
     1201 attcaaaaga tcttttaaga aggagatata ctgatggcaa atttcgagga ctggtgtgat
     1261 tcgacagagc gtaatatcag cgaccattat ttgcagtcaa taacggccag ggatgccgag
     1321 tgtatgttcg gcgttcaggt tatggccgcg ctaatacctg aacactatgc gtcaccaaga
     1381 aatatcgcta acgcatttga agctctaggt aaaccggggc tggcggcata cattgctgga
     1441 aaattgcctg aaacgaaaca gattcgttcc ggtgatttgg gtgagatatt tgcaactgaa
     1501 tggataaatg cacgtagcaa tggctataaa acgcctataa aaagattgcg ctggaaagat
     1561 catcgcaata tgtcgatgcg tggtgaggac gtcattggta tttacataga tcagtcttct
     1621 caacaacttt ttttccttaa aaccgcggcg gcgagtcgcg ccaaaatgac gggagaggtg
     1681 gtttctgaag cccgcgataa cttgaataaa gagcaagggc tgccatcttc acatgcccta
     1741 atgttcatag cagatcgcct gaatgaacag ggtgaggagc ttttggctaa agcgatctta
     1801 aatgcaacgc ttcggcaagg aattgttcca ggatgcgtcc ggcatcttat ctttctatta
     1861 tcgggcaact cttcagaaac tatgctgacc acttcaattg aaaagtatac aggtcaaaat
     1921 aatcagtggg gtgtttgtct aagaattgct cgccatggtg agtttattgc cgcaactttt
     1981 gaaaaggtga ttagcgatgc cagcaacagc tgatgaaatt atcgaagcta ttaaggaagc
     2041 ttcagctgta ggttttcgcg ggcgcctgat tgcacgcggg caagcccggt cggtgatctg
     2101 gcgagacggt gacttgccgc cggatgcacc ggagtttagc gcactgctca gtcaggacct
     2161 tcaaggctat gcttatgccc taatcgatct cggccttcgt cttcgagaac ttaatggaga
     2221 cgacgcctat gcgcgaattg cttttgaaca ggccggaacc gcactcgagt cggctattgc
     2281 taaaggaaaa cgcgacagcc gggatacaga ttttcacttc gtaatggctg ctgcaagcta
     2341 tcaccttgcg catctttcag cacgagccta ttcgttactc gcaatggttg gtcaggatga
     2401 taatttttca ccaatagaga gggctcttac acaactgatc cggcgtgatt tgcgtactct
     2461 gcgcgataat gcgttgggct ttcggctcag aggtgatggc agtgacgtga aaattactga
     2521 aatcctccag gctcgcctaa acttgccgca agacgagaac ggtgacagtg agtcagagga
     2581 ggacatcctt tttgatgggc ttgatttggc actgactgac gcctatatgt cggccatttc
     2641 tctctacctt ctcgctgtag aaaggggaga atcacgtttg ctttccaggg ctattgaaaa
     2701 attaagaatt agtctaagta tatgtgctca gtttaatatg ctaccccagt ggtggctaaa
     2761 ctttattacc attcatctgt tatctgacct ttggtcagat acattccatg aaagattgcc
     2821 actggttcca gttggtggtg acgctgctga atggcccgcg cttcgggaac tgtttattgc
     2881 actgcttcaa agacgacctc gagccgaaat tgatctctgg ccatcgcaga gagaggccgc
     2941 cggacgctct gttaatgaca atgacgattt ggttgtttcg ttgccaacca gcgctggaaa
     3001 aactcgtatt gctgaattgt gtattcttcg ctgtctggcg ggtggcaagc gtgttgtttt
     3061 catcaccccg cttcgtgcgc tttctgcaca aacagaagcc actctctcca gaacatttgg
     3121 gccgcttggg aaaacaatat caatgctgta tggcagcatt ggcgtaagtg gtatggatga
     3181 ggatgcaatt cgccagcgtg acatcgtcgt cgccacgcct gaaaaacttg actttgctct
     3241 ccggaatgac ccgtcaatta ttaatgatgt aggacttttt atttttgatg aggggcacat
     3301 gattggggcc gatgaacgag aggttcggta tgaagttcaa attcaacggc tgttgcgcag
     3361 acaggatgcc gatacgcgca gaattgtctg cctctcggca attctacctg atggtgagca
     3421 actcgatgat tttgccggtt ggctccgtcg tgacaagccc ggaggaccaa tcaaaaataa
     3481 ttggcgaccg acccgcttgc agtttggcga ggtcatatgg tccgctccag caggtcggct
     3541 taatctcagc gtcggttatg aggcggcttg ggtatcgcgt tttatcgtca gcagacaacc
     3601 gccaaaggtt aaattaccta ataaaaagca gcgaacaaaa atgtttccct ctgataacaa
     3661 ggagctctgc ttagcgacag cctggcgtct aattgaagat ggacagaccg tacttatcta
     3721 ctgcccactt cgccggagcg tcgaaccgtt tgccgagaca attgtcgatc tgcatcagcg
     3781 cggactactg ccatctttat ttgatgctgc cccggacatt ctggatacgg ctatctcgct
     3841 gggagaagaa tggttggggg cacacagccc gatacttgcc tgcctacggt tgggcgtggc
     3901 tcttcatcat ggcgcattgc ctacggctta ccgcaaagaa atcgaacgtt tgctgcgaga
     3961 tggggtactt aaagtgacga tctcatcacc gacgctagca caaggtctta atctttcggc
     4021 aacagctatc gtcatgcatt ctcttcatcg gaatcgcgag cttatcaaag tgtccgagtt
     4081 cagaaacgtc ataggcagag cagggcgcgc atatgtagat gttgaagggc tggtgattta
     4141 tccaatattc gataaagtaa ataaacgcca gactaactgg cataccctca catctgacac
     4201 gggggcccgt gagatggaga gcggactaat acagcttgtc tgtgtgttat tgattcgcat
     4261 gcatacacgg cttggtgggg atttaaaagc actcactgaa tatgtgacca acaacgccgt
     4321 agcgtgggaa tttccagaaa ttatgaccga atctccgcaa gaaagagata ttgcgcaggc
     4381 catctgggaa aaacaacttt caacgcttga tactgctatt ctgagccttc ttggtgagaa
     4441 tgatatcccg gatgaccaga ttgaaactgc tcttgatgac attcttcagt cttcactttg
     4501 gcaacgcagt ttgcagcgtt atagggatga gaatgaacgg attttgctga aatcgggact
     4561 cctgtcccgc agccgctaca tctggcagcg ctcaacggca gcagggcgcc gggggtattt
     4621 cctgtcgggg gtaggactta caaccgggct acgacttgat gctattgccg ctaaggccaa
     4681 ccaactattg attgatgcga atgcggctat aatgggcggt gatgccgaag aggcaattgc
     4741 tgctatcacg gcgctcgctg aagaggtctt taccttctat ccgtttatac ccgatccatt
     4801 gcctggcgac tggagaggta ttttacgttc ctggctattg ggtgaaccca tgactaacgt
     4861 cgcgaatacg caggcgtctg aaacgcttca attcgtcgag aatgggctgg tgtatcgact
     4921 tccgtgggca atggaggcta ttagggtacg tgcaacagct aatggagacc tcataggtga
     4981 tacagatacc acgctagacg attatgagtt aggttttgcg gttgctgctg ttgaaacagg
     5041 taccttgagc cgttcgtctt cactgcttat tcaggcgggt tttagctcta ggcttgcagc
     5101 cataaaagtg gttacagaca ctactgccga ttttcaatcc ggccaggagc ttagaaggtg
     5161 gctgaactct gaagaagtga taagccacac ggataatcat gactggccta cacctgaaac
     5221 cagagtgatg tggctggaat tcttgggtag tctcagcccc aaaggcagcc aggtttggag
     5281 tcgtcatcgt tataacggaa tggtggactg gagagatact ccggcagtga tcggcacacc
     5341 attgcagctt tacactgttg atggtattca tcacgtactg gctgatgatg gcactcctct
     5401 gggaagtata aatggcagaa ttaacacaaa cagaagaggt cttcttagag ttgaggtgga
     5461 tgatgagaat ggtcgtgcca tgttcgatta tttaggaccg gacgacttta tttctacctg
     5521 aaggaggact ttggcaggtg accaaaaaaa aacgcccggc ggcaaccgag cgttctgaac
     5581 aaatccagat ggagttctga ggtcattact ggatctatca acaggagtcc aagcgagctc
     5641 gatatcaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat
     5701 tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag
     5761 caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga agaagttgtc
     5821 catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg ctgaaacgaa
     5881 aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac
     5941 atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga
     6001 tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat
     6061 caccagctca ccgtctttca ttgccatacg aaattccgga tgagcattca tcaggcgggc
     6121 aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa
     6181 ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc
     6241 ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt
     6301 tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg
     6361 tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgcccga tcaagcgcta
     6421 gcggagtgta tactggctta ctatgttggc actgatgagg gtgtcagtga agtgcttcat
     6481 gtggcaggag aaaaaaggct gcaccggtgc gtcagcagaa tatgtgatac aggatatatt
     6541 ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg
     6601 cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga
     6661 gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat
     6721 ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6781 ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc
     6841 gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc
     6901 gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg
     6961 gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca
     7021 ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa
     7081 aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta
     7141 gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag
     7201 attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc
     7261 t
//
