LOCUS       sequence-358072-        7174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET based vector for protein expression in E.coli for targets fused 
            with C-terminal His.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7174)
  AUTHORS   Bruni R
  TITLE     Bruni Lab Empty Backbones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7174)
  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            "Unpublished"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: pC28-LIC-His
FEATURES             Location/Qualifiers
     source          1..7174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(41..60)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     201..223
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(217..408)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1217..2299)
                     /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     2261..2280
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2300..2377)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2583..2602
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2686..2705
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2686..2704
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2705..2729
                     /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             2744..2766
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(3219..3305,3306..4640)
                     /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             4695..4712
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4724..4741
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4794..4812)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4808..4855
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4892..5347
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4979..4998)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5189..5210
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5439..6254)
                     /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(5439..6254)
                     /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     6162..6181
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6246..6265
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6376..6964
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6865..6884
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7118..7135
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(7150..7174,1..115)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
       61 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
      121 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
      181 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
      241 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
      301 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
      361 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
      421 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
      481 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
      541 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
      601 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
      661 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
      721 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
      781 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
      841 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
      901 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
      961 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     1021 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     1081 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     1141 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     1201 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1261 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     1321 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     1381 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     1441 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     1501 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     1561 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     1621 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     1681 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     1741 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     1801 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     1861 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     1921 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     1981 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     2041 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     2101 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     2161 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     2221 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     2281 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     2341 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     2401 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     2461 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     2521 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     2581 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     2641 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
     2701 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
     2761 aggagatata ctatgcaggt cgttcactat tattatattt agtgaaatga gatattatga
     2821 tattttctga attgtgacta aaaaggcaac tttatgccca tgcaacagaa actataaaaa
     2881 atacagagaa tgaaaagaaa cagatagatt ttttagttct ttaggcccgt agtctgcaaa
     2941 tccttttatg attttctatc aaacaaaaga ggaaaataga ccagttgcaa tccaaacgag
     3001 agtctaatag aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat aaagcaggca
     3061 agacctaaaa tgtgtaaagg gcaaagtgta tactttggcg tcacccctta catattttag
     3121 gtcttttttt attgtgcgta actaacttgc catcttcaaa caggagggct ggaagaagca
     3181 gaccgctaac acagtacata aaaaaggaga catgaacgat gaacatcaaa aagtttgcaa
     3241 aacaagcaac agtattaacc tttactaccg cactgctggc aggaggcgca actcaagcgt
     3301 ttgcgaaaga aacgaaccaa aagccatata aggaaacata cggcatttcc catattacac
     3361 gccatgatat gctgcaaatc cctgaacagc aaaaaaatga aaaatataaa gttcctgagt
     3421 tcgattcgtc cacaattaaa aatatctctt ctgcaaaagg cctggacgtt tgggacagct
     3481 ggccattaca aaacactgac ggcactgtcg caaactatca cggctaccac atcgtctttg
     3541 cattagccgg agatcctaaa aatgcggatg acacatcgat ttacatgttc tatcaaaaag
     3601 tcggcgaaac ttctattgac agctggaaaa acgctggccg cgtctttaaa gacagcgaca
     3661 aattcgatgc aaatgattct atcctaaaag accaaacaca agaatggtca ggttcagcca
     3721 catttacatc tgacggaaaa atccgtttat tctacactga tttctccggt aaacattacg
     3781 gcaaacaaac actgacaact gcacaagtta acgtatcagc atcagacagc tctttgaaca
     3841 tcaacggtgt agaggattat aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg
     3901 tacagcagtt catcgatgaa ggcaactaca gctcaggcga caaccatacg ctgagagatc
     3961 ctcactacgt agaagataaa ggccacaaat acttagtatt tgaagcaaac actggaactg
     4021 aagatggcta ccaaggcgaa gaatctttat ttaacaaagc atactatggc aaaagcacat
     4081 cattcttccg tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc acggctgagt
     4141 tagcaaacgg cgctctcggt atgattgagc taaacgatga ttacacactg aaaaaagtga
     4201 tgaaaccgct gattgcatct aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta
     4261 aaatgaacgg caaatggtac ctgttcactg actcccgcgg atcaaaaatg acgattgacg
     4321 gcattacgtc taacgatatt tacatgcttg gttatgtttc taattcttta actggcccat
     4381 acaagccgct gaacaaaact ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa
     4441 cctttactta ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa
     4501 gctatatgac aaacagagga ttctacgcag acaaacaatc aacgtttgcg cctagcttcc
     4561 tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac
     4621 aattaacagt taacaaataa aaacgcaaaa gaaaatttta gtaagccacc tgcgccgaaa
     4681 acctctattt tcagcaccat catcatcacc attaaagctc gagcaccacc accaccacca
     4741 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     4801 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     4861 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     4921 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4981 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5041 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5101 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5161 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5221 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5281 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     5341 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5401 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     5461 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     5521 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     5581 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     5641 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     5701 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     5761 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     5821 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     5881 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     5941 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     6001 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     6061 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     6121 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     6181 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     6241 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     6301 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6361 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6421 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6481 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     6541 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6601 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6661 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6721 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6781 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6841 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6901 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6961 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7021 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     7081 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     7141 ggaagagcgc ctgatgcggt attttctcct tacg
//
