LOCUS       sequence-442983-        9491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 protein expression of MBP-His-TEV-ECOR31 Hachiman HamB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9491)
  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 9491)
  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: pBA1445_ECOR31HamB_purification
FEATURES             Location/Qualifiers
     source          1..9491
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             109..131
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             151..180
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             190..1290
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1264..1287
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1345..1365
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5022..5040)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5036..5083
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5449..5454,5455..5471,5472..5477))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5504..5522)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5590..5694
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5695..5763,5764..6555)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5913..5932)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6726..7314
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7215..7234
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7468..7485
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7500..7639
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7565..7584)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7725..7747
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7741..7932)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     9390..9409
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagtgccgg ctccggagag
       61 ctctttaatt aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt
      121 taagaaggag atatacatat gaaatcttct caccatcacc atcaccatca ccatcaccat
      181 ggttcttcta tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
      241 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
      301 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
      361 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
      421 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
      481 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
      541 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
      601 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
      661 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
      721 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
      781 gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc
      841 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
      901 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
      961 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     1021 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     1081 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     1141 attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     1201 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     1261 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac
     1321 aataacaaca acctcgggat cgaggaaaac ctgtacttcc aatccaatgc aatgccagca
     1381 acagctgatg aaattatcga agctattaag gaagcttcag ctgtaggttt tcgcgggcgc
     1441 ctgattgcac gcgggcaagc ccggtcggtg atctggcgag acggtgactt gccgccggat
     1501 gcaccggagt ttagcgcact gctcagtcag gaccttcaag gctatgctta tgccctaatc
     1561 gatctcggcc ttcgtcttcg agaacttaat ggagacgacg cctatgcgcg aattgctttt
     1621 gaacaggccg gaaccgcact cgagtcggct attgctaaag gaaaacgcga cagccgggat
     1681 acagattttc acttcgtaat ggctgctgca agctatcacc ttgcgcatct ttcagcacga
     1741 gcctattcgt tactcgcaat ggttggtcag gatgataatt tttcaccaat agagagggct
     1801 cttacacaac tgatccggcg tgatttgcgt actctgcgcg ataatgcgtt gggctttcgg
     1861 ctcagaggtg atggcagtga cgtgaaaatt actgaaatcc tccaggctcg cctaaacttg
     1921 ccgcaagacg agaacggtga cagtgagtca gaggaggaca tcctttttga tgggcttgat
     1981 ttggcactga ctgacgccta tatgtcggcc atttctctct accttctcgc tgtagaaagg
     2041 ggagaatcac gtttgctttc cagggctatt gaaaaattaa gaattagtct aagtatatgt
     2101 gctcagttta atatgctacc ccagtggtgg ctaaacttta ttaccattca tctgttatct
     2161 gacctttggt cagatacatt ccatgaaaga ttgccactgg ttccagttgg tggtgacgct
     2221 gctgaatggc ccgcgcttcg ggaactgttt attgcactgc ttcaaagacg acctcgagcc
     2281 gaaattgatc tctggccatc gcagagagag gccgccggac gctctgttaa tgacaatgac
     2341 gatttggttg tttcgttgcc aaccagcgct ggaaaaactc gtattgctga attgtgtatt
     2401 cttcgctgtc tggcgggtgg caagcgtgtt gttttcatca ccccgcttcg tgcgctttct
     2461 gcacaaacag aagccactct ctccagaaca tttgggccgc ttgggaaaac aatatcaatg
     2521 ctgtatggca gcattggcgt aagtggtatg gatgaggatg caattcgcca gcgtgacatc
     2581 gtcgtcgcca cgcctgaaaa acttgacttt gctctccgga atgacccgtc aattattaat
     2641 gatgtaggac tttttatttt tgatgagggg cacatgattg gggccgatga acgagaggtt
     2701 cggtatgaag ttcaaattca acggctgttg cgcagacagg atgccgatac gcgcagaatt
     2761 gtctgcctct cggcaattct acctgatggt gagcaactcg atgattttgc cggttggctc
     2821 cgtcgtgaca agcccggagg accaatcaaa aataattggc gaccgacccg cttgcagttt
     2881 ggcgaggtca tatggtccgc tccagcaggt cggcttaatc tcagcgtcgg ttatgaggcg
     2941 gcttgggtat cgcgttttat cgtcagcaga caaccgccaa aggttaaatt acctaataaa
     3001 aagcagcgaa caaaaatgtt tccctctgat aacaaggagc tctgcttagc gacagcctgg
     3061 cgtctaattg aagatggaca gaccgtactt atctactgcc cacttcgccg gagcgtcgaa
     3121 ccgtttgccg agacaattgt cgatctgcat cagcgcggac tactgccatc tttatttgat
     3181 gctgccccgg acattctgga tacggctatc tcgctgggag aagaatggtt gggggcacac
     3241 agcccgatac ttgcctgcct acggttgggc gtggctcttc atcatggcgc attgcctacg
     3301 gcttaccgca aagaaatcga acgtttgctg cgagatgggg tacttaaagt gacgatctca
     3361 tcaccgacgc tagcacaagg tcttaatctt tcggcaacag ctatcgtcat gcattctctt
     3421 catcggaatc gcgagcttat caaagtgtcc gagttcagaa acgtcatagg cagagcaggg
     3481 cgcgcatatg tagatgttga agggctggtg atttatccaa tattcgataa agtaaataaa
     3541 cgccagacta actggcatac cctcacatct gacacggggg cccgtgagat ggagagcgga
     3601 ctaatacagc ttgtctgtgt gttattgatt cgcatgcata cacggcttgg tggggattta
     3661 aaagcactca ctgaatatgt gaccaacaac gccgtagcgt gggaatttcc agaaattatg
     3721 accgaatctc cgcaagaaag agatattgcg caggccatct gggaaaaaca actttcaacg
     3781 cttgatactg ctattctgag ccttcttggt gagaatgata tcccggatga ccagattgaa
     3841 actgctcttg atgacattct tcagtcttca ctttggcaac gcagtttgca gcgttatagg
     3901 gatgagaatg aacggatttt gctgaaatcg ggactcctgt cccgcagccg ctacatctgg
     3961 cagcgctcaa cggcagcagg gcgccggggg tatttcctgt cgggggtagg acttacaacc
     4021 gggctacgac ttgatgctat tgccgctaag gccaaccaac tattgattga tgcgaatgcg
     4081 gctataatgg gcggtgatgc cgaagaggca attgctgcta tcacggcgct cgctgaagag
     4141 gtctttacct tctatccgtt tatacccgat ccattgcctg gcgactggag aggtatttta
     4201 cgttcctggc tattgggtga acccatgact aacgtcgcga atacgcaggc gtctgaaacg
     4261 cttcaattcg tcgagaatgg gctggtgtat cgacttccgt gggcaatgga ggctattagg
     4321 gtacgtgcaa cagctaatgg agacctcata ggtgatacag ataccacgct agacgattat
     4381 gagttaggtt ttgcggttgc tgctgttgaa acaggtacct tgagccgttc gtcttcactg
     4441 cttattcagg cgggttttag ctctaggctt gcagccataa aagtggttac agacactact
     4501 gccgattttc aatccggcca ggagcttaga aggtggctga actctgaaga agtgataagc
     4561 cacacggata atcatgactg gcctacacct gaaaccagag tgatgtggct ggaattcttg
     4621 ggtagtctca gccccaaagg cagccaggtt tggagtcgtc atcgttataa cggaatggtg
     4681 gactggagag atactccggc agtgatcggc acaccattgc agctttacac tgttgatggt
     4741 attcatcacg tactggctga tgatggcact cctctgggaa gtataaatgg cagaattaac
     4801 acaaacagaa gaggtcttct tagagttgag gtggatgatg agaatggtcg tgccatgttc
     4861 gattatttag gaccggacga ctttatttct acctaacaac attggaagtg gataacggat
     4921 ccgcgatcgc ggcgcgccac ctggtggccg gccggtacca cgcgtgcgcg ctgatccggc
     4981 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     5041 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     5101 atccggatat ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata gtggctccaa
     5161 gtagcgaagc gagcaggact gggcggcggc caaagcggtc ggacagtgct ccgagaacgg
     5221 gtgcgcatag aaattgcatc aacgcatata gcgctagcag cacgccatag tgactggcga
     5281 tgctgtcgga atggacgata tcccgcaaga ggcccggcag taccggcata accaagccta
     5341 tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca
     5401 tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat
     5461 cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta
     5521 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5581 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5641 ctcatgagac aataaccctg ataaatgctt caataacatt gaaaaaggaa gagtatgagt
     5701 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5761 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5821 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5881 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     5941 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6001 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6061 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6121 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6181 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     6241 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6301 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6361 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6421 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6481 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6541 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6601 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6661 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6721 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6781 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6841 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     6901 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6961 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7021 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7081 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7141 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7201 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7261 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7321 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7381 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7441 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     7501 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     7561 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     7621 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     7681 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     7741 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     7801 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     7861 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     7921 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     7981 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     8041 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     8101 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     8161 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     8221 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     8281 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     8341 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     8401 ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat
     8461 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc
     8521 tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag
     8581 gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg
     8641 cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg
     8701 atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt
     8761 ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg
     8821 ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc
     8881 agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg
     8941 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     9001 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     9061 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     9121 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     9181 aagggcatcg gtcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     9241 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     9301 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     9361 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     9421 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     9481 atcccgcgaa a
//
