LOCUS       sequence-251071-        6988 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 6988)
  TITLE     Cloning Vectors
REFERENCE   2  (bases 1 to 6988)
  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: pCW-LIC
FEATURES             Location/Qualifiers
     source          1..6988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(32..37,38..55,56..62)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    70..86
                     /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)."
     promoter        join(141..146,147..162,163..169)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    177..193
                     /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)."
     promoter        join(236..241,242..257,258..264)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    272..288
                     /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)."
     protein_bind    complement(361..394)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        409..854
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(855..941,942..2276)
                     /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"
     primer_bind     complement(2448..2466)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      2528..2983
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2615..2634)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2825..2846
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3016..3120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3121..3189,3190..3981)
                     /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(3339..3358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4152..4740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4641..4660
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4894..4911
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4926..5067
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4993..5012)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5153..5175
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5188..5649)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVLPRAFR"
     primer_bind     5598..5620
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5612..5629
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5613..5629
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5645..5661)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5645..5661)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5669..5685
                     /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)."
     promoter        complement(join(5693..5699,5700..5717,5718..5723))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             complement(5772..6854)
                     /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     6816..6835
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6855..6932)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
ORIGIN
        1 taagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa
       61 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa caggatcgat ccatcgatga
      121 gcttactccc catccccctg ttgacaatta atcatcggct cgtataatgt gtggaattgt
      181 gagcggataa caatttcaca caggaaacag gatcagctta ctccccatcc ccctgttgac
      241 aattaatcat cggctcgtat aatgtgtgga attgtgagcg gataacaatt tcacacagga
      301 aacaggatcc atcgatgctt aggaggtcat atgattatga gttctcctcc tgaaagatcc
      361 ataacttcgt atagcataca ttatacgaag ttatgcggcc gcgacgtcca catatacctg
      421 ccgttcacta ttatttagtg aaatgagata ttatgatatt ttctgaattg tgattaaaaa
      481 ggcaacttta tgcccatgca acagaaacta taaaaaatac agagaatgaa aagaaacaga
      541 tagatttttt agttctttag gcccgtagtc tgcaaatcct tttatgattt tctatcaaac
      601 aaaagaggaa aatagaccag ttgcaatcca aacgagagtc taatagaatg aggtcgaaaa
      661 gtaaatcgcg cgggtttgtt actgataaag caggcaagac ctaaaatgtg taaagggcaa
      721 agtgtatact ttggcgtcac cccttacata ttttaggtct ttttttattg tgcgtaacta
      781 acttgccatc ttcaaacagg agggctggaa gaagcagacc gctaacacag tacataaaaa
      841 aggagacatg aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta ttaaccttta
      901 ctaccgcact gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg aaccaaaagc
      961 catataagga aacatacggc atttcccata ttacacgcca tgatatgctg caaatccctg
     1021 aacagcaaaa aaatgaaaaa tataaagttc ctgagttcga ttcgtccaca attaaaaata
     1081 tctcttctgc aaaaggcctg gacgtttggg acagctggcc attacaaaac actgacggca
     1141 ctgtcgcaaa ctatcacggc taccacatcg tctttgcatt agccggagat cctaaaaatg
     1201 cggatgacac atcgatttac atgttctatc aaaaagtcgg cgaaacttct attgacagct
     1261 ggaaaaacgc tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat gattctatcc
     1321 taaaagacca aacacaagaa tggtcaggtt cagccacatt tacatctgac ggaaaaatcc
     1381 gtttattcta cactgatttc tccggtaaac attacggcaa acaaacactg acaactgcac
     1441 aagttaacgt atcagcatca gacagctctt tgaacatcaa cggtgtagag gattataaat
     1501 caatctttga cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc gatgaaggca
     1561 actacagctc aggcgacaac catacgctga gagatcctca ctacgtagaa gataaaggcc
     1621 acaaatactt agtatttgaa gcaaacactg gaactgaaga tggctaccaa ggcgaagaat
     1681 ctttatttaa caaagcatac tatggcaaaa gcacatcatt cttccgtcaa gaaagtcaaa
     1741 aacttctgca aagcgataaa aaacgcacgg ctgagttagc aaacggcgct ctcggtatga
     1801 ttgagctaaa cgatgattac acactgaaaa aagtgatgaa accgctgatt gcatctaaca
     1861 cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa tggtacctgt
     1921 tcactgactc ccgcggatca aaaatgacga ttgacggcat tacgtctaac gatatttaca
     1981 tgcttggtta tgtttctaat tctttaactg gcccatacaa gccgctgaac aaaactggcc
     2041 ttgtgttaaa aatggatctt gatcctaacg atgtaacctt tacttactca cacttcgctg
     2101 tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac agaggattct
     2161 acgcagacaa acaatcaacg tttgcgccta gcttcctgct gaacatcaaa ggcaagaaaa
     2221 catctgttgt caaagacagc atccttgaac aaggacaatt aacagttaac aaataaaaac
     2281 gcaaaagaaa atgccgatat cctattggca ttgacgtcag gtggcacttt tcgaggagat
     2341 catgcacatg atgacgaagc ttatcgctga taagctgtca aacatgagca gatctgagcc
     2401 cgcctaatga gcgggctttt ttttcagatc tgcttgaaga cgaaagggcc tcgtgatacg
     2461 cctattttta taggttaatg tcatgataat aatggtttct tagacgatgc gtcaaagcaa
     2521 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2581 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2641 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2701 cgatttagag ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     2761 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2821 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt
     2881 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2941 aaatttaacg cgaattttaa caaaatatta acgtttacaa tttcatcgtc aggtggcact
     3001 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3061 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3121 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3181 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3241 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3301 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3361 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3421 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3481 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3541 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3601 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3661 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3721 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3781 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3841 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3901 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3961 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4021 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4081 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4141 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4201 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4261 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4321 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4381 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4441 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4501 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4561 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4621 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4681 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4741 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4801 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4861 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4921 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataatg
     4981 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     5041 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     5101 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5161 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagaa cgccatcaaa
     5221 aataattcgc gtctggcctt cctgtagcca gctttcatca acattaaatg tgagcgagta
     5281 acaacccgtc ggattctccg tgggaacaaa cggcggattg accgtaatgg gataggtcac
     5341 gttggtgtag atgggcgcat cgtaaccgtg catctgccag tttgagggga cgacgacagt
     5401 atcggcctca ggaagatcgc actccagcca gctttccggc accgcttctg gtgccggaaa
     5461 ccaggcaaag cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     5521 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     5581 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aatccgtaat
     5641 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacattatac
     5701 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa cttacattaa
     5761 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5821 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     5881 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     5941 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     6001 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     6061 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     6121 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     6181 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     6241 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     6301 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     6361 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     6421 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     6481 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     6541 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     6601 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     6661 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     6721 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     6781 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     6841 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     6901 ccgcgaaagg ttttgcacca ttcgatggtg tcctggcacg acaggtttcc cgactggaaa
     6961 gcgggcagtg agcgcaacgc aattaatg
//
