LOCUS       sequence-319519-        2641 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Bacterial expression plasmid with sacB gene. Suitable for LIC 
            cloning of a gene insert replacing the sacB..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2641)
  TITLE     pET32a-Trx-His6-TEV_SacB
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 2641)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pET32a-Trx-His6-TEV_SacB
FEATURES             Location/Qualifiers
     source          1..2641
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(join(234..1568,1569..1655))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        complement(1656..2101)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             complement(2117..2137)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(2162..2179)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2201..2527)
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     RBS             2535..2557
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2572..2596
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(2596..2615)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2597..2615)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag caagcttgtc gacggagctc gaattcggat ccgtatccac
      181 ctttactgga gaccgtcaat gccaatagga tatcggcatt ttcttttgcg tttttatttg
      241 ttaactgtta attgtccttg ttcaaggatg ctgtctttga caacagatgt tttcttgcct
      301 ttgatgttca gcaggaagct aggcgcaaac gttgattgtt tgtctgcgta gaatcctctg
      361 tttgtcatat agcttgtaat cacgacattg tttcctttcg cttgaggtac agcgaagtgt
      421 gagtaagtaa aggttacatc gttaggatca agatccattt ttaacacaag gccagttttg
      481 ttcagcggct tgtatgggcc agttaaagaa ttagaaacat aaccaagcat gtaaatatcg
      541 ttagacgtaa tgccgtcaat cgtcattttt gatccgcggg agtcagtgaa caggtaccat
      601 ttgccgttca ttttaaagac gttcgcgcgt tcaatttcat ctgttactgt gttagatgca
      661 atcagcggtt tcatcacttt tttcagtgtg taatcatcgt ttagctcaat cataccgaga
      721 gcgccgtttg ctaactcagc cgtgcgtttt ttatcgcttt gcagaagttt ttgactttct
      781 tgacggaaga atgatgtgct tttgccatag tatgctttgt taaataaaga ttcttcgcct
      841 tggtagccat cttcagttcc agtgtttgct tcaaatacta agtatttgtg gcctttatct
      901 tctacgtagt gaggatctct cagcgtatgg ttgtcgcctg agctgtagtt gccttcatcg
      961 atgaactgct gtacattttg atacgttttt ccgtcaccgt caaagattga tttataatcc
     1021 tctacaccgt tgatgttcaa agagctgtct gatgctgata cgttaacttg tgcagttgtc
     1081 agtgtttgtt tgccgtaatg tttaccggag aaatcagtgt agaataaacg gatttttccg
     1141 tcagatgtaa atgtggctga acctgaccat tcttgtgttt ggtcttttag gatagaatca
     1201 tttgcatcga atttgtcgct gtctttaaag acgcggccag cgtttttcca gctgtcaata
     1261 gaagtttcgc cgactttttg atagaacatg taaatcgatg tgtcatccgc atttttagga
     1321 tctccggcta atgcaaagac gatgtggtag ccgtgatagt ttgcgacagt gccgtcagtg
     1381 ttttgtaatg gccagctgtc ccaaacgtcc aggccttttg cagaagagat atttttaatt
     1441 gtggacgaat cgaactcagg aactttatat ttttcatttt tttgctgttc agggatttgc
     1501 agcatatcat ggcgtgtaat atgggaaatg ccgtatgttt ccttatatgg cttttggttc
     1561 gtttctttcg caaacgcttg agttgcgcct cctgccagca gtgcggtagt aaaggttaat
     1621 actgttgctt gttttgcaaa ctttttgatg ttcatcgttc atgtctcctt ttttatgtac
     1681 tgtgttagcg gtctgcttct tccagccctc ctgtttgaag atggcaagtt agttacgcac
     1741 aataaaaaaa gacctaaaat atgtaagggg tgacgccaaa gtatacactt tgccctttac
     1801 acattttagg tcttgcctgc tttatcagta acaaacccgc gcgatttact tttcgacctc
     1861 attctattag actctcgttt ggattgcaac tggtctattt tcctcttttg tttgatagaa
     1921 aatcataaaa ggatttgcag actacgggcc taaagaacta aaaaatctat ctgtttcttt
     1981 tcattctctg tattttttat agtttctgtt gcatgggcat aaagttgcct ttttaatcac
     2041 aattcagaaa atatcataat atctcatttc actaaataat agtgaacggc aggtatatgt
     2101 ggacgtcggt ctccatggat tggaagtaca ggttctcggt acccagatct acaccagaag
     2161 aatgatgatg atgatggtgc atatggccag aaccagaacc ggccaggtta gcgtcgagga
     2221 actctttcaa ctgaccttta gacagtgcac ccactttggt tgccgccact tcaccgtttt
     2281 tgaacagcag cagagtcggg ataccacgga tgccatattt cggcgcagtg ccagggtttt
     2341 gatcgatgtt cagttttgca acggtcagtt tgccctgata ttcgtcagcg atttcatcca
     2401 gaatcggggc gatcattttg cacggaccgc accactctgc ccagaaatcg acgaggatcg
     2461 ccccgtccgc tttgagtaca tccgtgtcaa aactgtcgtc agtcaggtga ataattttat
     2521 cgctcatatg tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt
     2581 atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga gatcgatctc
     2641 g
//
