LOCUS       sequence-337962-        7731 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7731)
  TITLE     pET32a-Trx-His6-TEV_SacB
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7731)
  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            "Unpublished"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..7731
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     primer_bind     complement(2703..2722)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2928..3005
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3006..4088
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3025..3044)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4897..5088
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5082..5104)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5190..5329
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5245..5264
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5344..5361)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5515..6103)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5595..5614)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6274..7065,7066..7134))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6897..6916
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7135..7239)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7265..7720)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7402..7423)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7614..7633
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
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 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     2701 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     2761 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     2821 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     2881 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
     2941 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     3001 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     3061 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     3121 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     3181 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     3241 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     3301 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     3361 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     3421 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     3481 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     3541 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     3601 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     3661 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     3721 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     3781 agtccgggct gcgcgttggt gcggacatct cggtagtggg atacgacgat accgaagaca
     3841 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     3901 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     3961 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     4021 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     4081 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
     4141 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     4201 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     4261 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     4321 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     4381 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc
     4441 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     4501 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     4561 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     4621 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
     4681 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
     4741 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
     4801 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
     4861 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
     4921 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
     4981 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
     5041 taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     5101 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     5161 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     5221 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt
     5281 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     5341 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5401 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5461 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5521 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5581 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5641 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5701 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5761 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5821 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5881 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5941 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6001 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6061 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6121 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6181 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6241 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6301 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6361 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6421 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6481 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6541 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt
     6601 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6661 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6721 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6781 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6841 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6901 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6961 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7021 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7081 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7141 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7201 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7261 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     7321 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     7381 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     7441 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     7501 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     7561 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     7621 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     7681 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc a
//
