LOCUS       sequence-246717-        7324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SGC Empty backbone for bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7324)
  TITLE     Cloning Vectors
REFERENCE   2  (bases 1 to 7324)
  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"
COMMENT     Sequence Label: pET28a-LIC
FEATURES             Location/Qualifiers
     source          1..7324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     206..225
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     309..328
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        309..327
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    328..352
                     /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)."
     RBS             367..389
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             408..425
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             435..452
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    complement(480..513)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        528..973
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(974..1060,1061..2395)
                     /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"
     CDS             2497..2514
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2567..2585)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2581..2628
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2665..3120
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2752..2771)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2962..2983
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3212..4027)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3212..4027)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3935..3954
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4019..4038
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4149..4737
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4638..4657
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4891..4908
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4923..5062
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4988..5007)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5148..5170
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5164..5355)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6164..7246)
                     /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     7208..7227
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7247..7324)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
       61 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
      121 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
      181 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      241 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      301 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
      361 aataattttg tttaacttta agaaggagat ataccatggg cagcagccat catcatcatc
      421 atcacagcag cggcctggtt ccgcgtggta gtattatgag ttctcctcct gaaagatcca
      481 taacttcgta tagcatacat tatacgaagt tatgcggccg cgacgtccac atatacctgc
      541 cgttcactat tatttagtga aatgagatat tatgatattt tctgaattgt gattaaaaag
      601 gcaactttat gcccatgcaa cagaaactat aaaaaataca gagaatgaaa agaaacagat
      661 agatttttta gttctttagg cccgtagtct gcaaatcctt ttatgatttt ctatcaaaca
      721 aaagaggaaa atagaccagt tgcaatccaa acgagagtct aatagaatga ggtcgaaaag
      781 taaatcgcgc gggtttgtta ctgataaagc aggcaagacc taaaatgtgt aaagggcaaa
      841 gtgtatactt tggcgtcacc ccttacatat tttaggtctt tttttattgt gcgtaactaa
      901 cttgccatct tcaaacagga gggctggaag aagcagaccg ctaacacagt acataaaaaa
      961 ggagacatga acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat taacctttac
     1021 taccgcactg ctggcaggag gcgcaactca agcgtttgcg aaagaaacga accaaaagcc
     1081 atataaggaa acatacggca tttcccatat tacacgccat gatatgctgc aaatccctga
     1141 acagcaaaaa aatgaaaaat ataaagttcc tgagttcgat tcgtccacaa ttaaaaatat
     1201 ctcttctgca aaaggcctgg acgtttggga cagctggcca ttacaaaaca ctgacggcac
     1261 tgtcgcaaac tatcacggct accacatcgt ctttgcatta gccggagatc ctaaaaatgc
     1321 ggatgacaca tcgatttaca tgttctatca aaaagtcggc gaaacttcta ttgacagctg
     1381 gaaaaacgct ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg attctatcct
     1441 aaaagaccaa acacaagaat ggtcaggttc agccacattt acatctgacg gaaaaatccg
     1501 tttattctac actgatttct ccggtaaaca ttacggcaaa caaacactga caactgcaca
     1561 agttaacgta tcagcatcag acagctcttt gaacatcaac ggtgtagagg attataaatc
     1621 aatctttgac ggtgacggaa aaacgtatca aaatgtacag cagttcatcg atgaaggcaa
     1681 ctacagctca ggcgacaacc atacgctgag agatcctcac tacgtagaag ataaaggcca
     1741 caaatactta gtatttgaag caaacactgg aactgaagat ggctaccaag gcgaagaatc
     1801 tttatttaac aaagcatact atggcaaaag cacatcattc ttccgtcaag aaagtcaaaa
     1861 acttctgcaa agcgataaaa aacgcacggc tgagttagca aacggcgctc tcggtatgat
     1921 tgagctaaac gatgattaca cactgaaaaa agtgatgaaa ccgctgattg catctaacac
     1981 agtaacagat gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat ggtacctgtt
     2041 cactgactcc cgcggatcaa aaatgacgat tgacggcatt acgtctaacg atatttacat
     2101 gcttggttat gtttctaatt ctttaactgg cccatacaag ccgctgaaca aaactggcct
     2161 tgtgttaaaa atggatcttg atcctaacga tgtaaccttt acttactcac acttcgctgt
     2221 acctcaagcg aaaggaaaca atgtcgtgat tacaagctat atgacaaaca gaggattcta
     2281 cgcagacaaa caatcaacgt ttgcgcctag cttcctgctg aacatcaaag gcaagaaaac
     2341 atctgttgtc aaagacagca tccttgaaca aggacaatta acagttaaca aataaaaacg
     2401 caaaagaaaa tgccgatatc ctattggcat tgacgtcagg tggcactttt cgaggagatc
     2461 atgcacatga tgacgaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
     2521 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2581 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2641 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2701 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2761 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2821 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2881 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2941 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3001 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3061 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3121 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3181 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     3241 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     3301 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3361 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3421 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     3481 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3541 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3601 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3661 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3721 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3781 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3841 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3901 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3961 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     4021 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     4081 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4141 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4201 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4261 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4321 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4381 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4441 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4501 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4561 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4621 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4681 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4741 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4801 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4861 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4921 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4981 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     5041 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     5101 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5161 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     5221 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     5281 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     5341 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     5401 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     5461 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     5521 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     5581 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5641 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5701 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     5761 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5821 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5881 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5941 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     6001 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     6061 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     6121 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     6181 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6241 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     6301 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     6361 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     6421 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     6481 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     6541 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     6601 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     6661 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     6721 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     6781 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     6841 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     6901 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     6961 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     7021 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     7081 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     7141 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     7201 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     7261 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     7321 tgtc
//
