LOCUS       sequence-442988-        6745 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 protein expression of MBP-His-TEV-ECOR31 Hachiman HamA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6745)
  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 6745)
  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: pBA1444_ECOR31HamA_purification
FEATURES             Location/Qualifiers
     source          1..6745
                     /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(2276..2294)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2290..2337
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2703..2708,2709..2725,2726..2731))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2758..2776)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2844..2948
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2949..3017,3018..3809)
                     /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(3167..3186)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3980..4568
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4469..4488
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4722..4739
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4754..4893
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4819..4838)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4979..5001
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4995..5186)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6644..6663
                     /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 aatggcaaat
     1381 ttcgaggact ggtgtgattc gacagagcgt aatatcagcg accattattt gcagtcaata
     1441 acggccaggg atgccgagtg tatgttcggc gttcaggtta tggccgcgct aatacctgaa
     1501 cactatgcgt caccaagaaa tatcgctaac gcatttgaag ctctaggtaa accggggctg
     1561 gcggcataca ttgctggaaa attgcctgaa acgaaacaga ttcgttccgg tgatttgggt
     1621 gagatatttg caactgaatg gataaatgca cgtagcaatg gctataaaac gcctataaaa
     1681 agattgcgct ggaaagatca tcgcaatatg tcgatgcgtg gtgaggacgt cattggtatt
     1741 tacatagatc agtcttctca acaacttttt ttccttaaaa ccgaagcgaa aagtcgcgcc
     1801 aaaatgacgg gagaggtggt ttctgaagcc cgcgataact tgaataaaga gcaagggctg
     1861 ccatcttcac atgccctaat gttcatagca gatcgcctga atgaacaggg tgaggagctt
     1921 ttggctaaag cgatcttaaa tgcaacgctt cggcaaggaa ttgttccagg atgcgtccgg
     1981 catcttatct ttctattatc gggcaactct tcagaaacta tgctgaccac ttcaattgaa
     2041 aagtatacag gtcaaaataa tcagtggggt gtttgtctaa gaattgctcg ccatggtgag
     2101 tttattgccg caacttttga aaaggtgatt agcgatgcca gcaacagcta ataacattgg
     2161 aatggataac ggatccgcga tcgcggcgcg ccacctggtg gccggccggt accacgcgtg
     2221 cgcgctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     2281 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2341 aaaggaggaa ctatatccgg atatccacag gacgggtgtg gtcgccatga tcgcgtagtc
     2401 gatagtggct ccaagtagcg aagcgagcag gactgggcgg cggccaaagc ggtcggacag
     2461 tgctccgaga acgggtgcgc atagaaattg catcaacgca tatagcgcta gcagcacgcc
     2521 atagtgactg gcgatgctgt cggaatggac gatatcccgc aagaggcccg gcagtaccgg
     2581 cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc
     2641 attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc
     2701 gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga cgaaagggcc
     2761 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     2821 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2881 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa cattgaaaaa
     2941 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3001 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3061 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3121 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3181 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3241 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3301 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3361 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3421 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3481 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3541 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3601 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3661 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3721 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3781 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3841 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3901 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3961 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4021 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4081 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4141 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4201 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4261 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4321 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4381 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4441 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4501 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4561 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4621 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4681 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4741 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4801 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4861 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4921 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4981 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     5041 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     5101 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     5161 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     5221 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     5281 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     5341 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     5401 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     5461 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     5521 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     5581 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     5641 atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag
     5701 atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc
     5761 aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt
     5821 ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa
     5881 ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat
     5941 aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg
     6001 atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg
     6061 cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc
     6121 gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg
     6181 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     6241 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     6301 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     6361 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     6421 ggttgaaggc tctcaagggc atcggtcgac gctctccctt atgcgactcc tgcattagga
     6481 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     6541 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     6601 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     6661 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     6721 ggatcgagat ctcgatcccg cgaaa
//
