LOCUS       sequence-12259-e        7446 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 7446)
  AUTHORS   Savitsky P, Bray J, Cooper CD, Marsden BD, Mahajan P, Burgess-Brown 
            NA, Gileadi O
  TITLE     High-throughput production of human proteins for crystallization: 
            the SGC experience.
  JOURNAL   J Struct Biol. 2010 Oct;172(1):3-13. doi: 10.1016/j.jsb.2010.06.008.
            Epub 2010 Jun 10.
  PUBMED    20541610
REFERENCE   2  (bases 1 to 7446)
  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"; doi: 
            "10.1016/j.jsb.2010.06.008"; journalName: "J Struct Biol"; date: 
            "2010-10"; volume: "172"; issue: "1"; pages: "3-13"
COMMENT     Sequence Label: pNIC-ZB
FEATURES             Location/Qualifiers
     source          1..7446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             119..289
                     /codon_start=1
                     /product="Z domain of staphylococcal protein A, engineered 
                     to contain multiple positive charges (Hedhammar and Hober, 
                     2007)"
                     /label=Z-basic tag
                     /note="useful for purifying tagged proteins by 
                     cation-exchange chromatography"
                     /translation="VDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLL
                     AEAKKLNDAQPK"
     CDS             296..316
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     promoter        332..777
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(778..864,865..2199)
                     /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             2307..2324
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2377..2395)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2391..2438
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2475..2930
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2562..2581)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2772..2793
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3023..3838)
                     /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(3023..3838)
                     /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     3746..3765
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3830..3849
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3960..4548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4449..4468
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4702..4719
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4734..4876
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4802..4821)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4962..4984
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4978..5169)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5978..7060)
                     /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     7022..7041
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7061..7138)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7344..7363
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 ggataacaag ttcaacaagg agcgtcgaag agctcgccgt gaaattcgcc atctgccgaa
      181 cctgaaccgc gaacagcgtc gcgcatttat tcgcagcctg cgcgatgatc cgagccagag
      241 cgcgaacctg ctggcggaag cgaagaagct gaacgatgcg cagccgaagg gtaccgagaa
      301 cctgtacttc caatccatgg agaccgacgt ccacatatac ctgccgttca ctattattta
      361 gtgaaatgag atattatgat attttctgaa ttgtgattaa aaaggcaact ttatgcccat
      421 gcaacagaaa ctataaaaaa tacagagaat gaaaagaaac agatagattt tttagttctt
      481 taggcccgta gtctgcaaat ccttttatga ttttctatca aacaaaagag gaaaatagac
      541 cagttgcaat ccaaacgaga gtctaataga atgaggtcga aaagtaaatc gcgcgggttt
      601 gttactgata aagcaggcaa gacctaaaat gtgtaaaggg caaagtgtat actttggcgt
      661 caccccttac atattttagg tcttttttta ttgtgcgtaa ctaacttgcc atcttcaaac
      721 aggagggctg gaagaagcag accgctaaca cagtacataa aaaaggagac atgaacgatg
      781 aacatcaaaa agtttgcaaa acaagcaaca gtattaacct ttactaccgc actgctggca
      841 ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa ggaaacatac
      901 ggcatttccc atattacacg ccatgatatg ctgcaaatcc ctgaacagca aaaaaatgaa
      961 aaatataaag ttcctgagtt cgattcgtcc acaattaaaa atatctcttc tgcaaaaggc
     1021 ctggacgttt gggacagctg gccattacaa aacactgacg gcactgtcgc aaactatcac
     1081 ggctaccaca tcgtctttgc attagccgga gatcctaaaa atgcggatga cacatcgatt
     1141 tacatgttct atcaaaaagt cggcgaaact tctattgaca gctggaaaaa cgctggccgc
     1201 gtctttaaag acagcgacaa attcgatgca aatgattcta tcctaaaaga ccaaacacaa
     1261 gaatggtcag gttcagccac atttacatct gacggaaaaa tccgtttatt ctacactgat
     1321 ttctccggta aacattacgg caaacaaaca ctgacaactg cacaagttaa cgtatcagca
     1381 tcagacagct ctttgaacat caacggtgta gaggattata aatcaatctt tgacggtgac
     1441 ggaaaaacgt atcaaaatgt acagcagttc atcgatgaag gcaactacag ctcaggcgac
     1501 aaccatacgc tgagagatcc tcactacgta gaagataaag gccacaaata cttagtattt
     1561 gaagcaaaca ctggaactga agatggctac caaggcgaag aatctttatt taacaaagca
     1621 tactatggca aaagcacatc attcttccgt caagaaagtc aaaaacttct gcaaagcgat
     1681 aaaaaacgca cggctgagtt agcaaacggc gctctcggta tgattgagct aaacgatgat
     1741 tacacactga aaaaagtgat gaaaccgctg attgcatcta acacagtaac agatgaaatt
     1801 gaacgcgcga acgtctttaa aatgaacggc aaatggtacc tgttcactga ctcccgcgga
     1861 tcaaaaatga cgattgacgg cattacgtct aacgatattt acatgcttgg ttatgtttct
     1921 aattctttaa ctggcccata caagccgctg aacaaaactg gccttgtgtt aaaaatggat
     1981 cttgatccta acgatgtaac ctttacttac tcacacttcg ctgtacctca agcgaaagga
     2041 aacaatgtcg tgattacaag ctatatgaca aacagaggat tctacgcaga caaacaatca
     2101 acgtttgcgc ctagcttcct gctgaacatc aaaggcaaga aaacatctgt tgtcaaagac
     2161 agcatccttg aacaaggaca attaacagtt aacaaataaa aacgcaaaag aaaatgccga
     2221 tatcctattg gcattgacgg tctccagtaa aggtggatac ggatccgaat tcgagctccg
     2281 tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta
     2341 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2401 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2461 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2521 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2581 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2641 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2701 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2761 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2821 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2881 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt caggtggcac
     2941 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3001 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     3061 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3121 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3181 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3241 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3301 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3361 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3421 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3481 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3541 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3601 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3661 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3721 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3781 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3841 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3901 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3961 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4021 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4081 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4141 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4201 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4261 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4321 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4381 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4441 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4501 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4561 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4621 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4681 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4741 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg tgcactctca
     4801 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4861 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4921 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4981 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     5041 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     5101 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     5161 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     5221 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     5281 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     5341 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     5401 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     5461 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     5521 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     5581 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     5641 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     5701 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     5761 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     5821 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     5881 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5941 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6001 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6061 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     6121 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     6181 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     6241 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     6301 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     6361 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     6421 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     6481 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     6541 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     6601 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     6661 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     6721 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     6781 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     6841 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     6901 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     6961 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     7021 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     7081 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     7141 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     7201 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     7261 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     7321 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     7381 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     7441 cgaaat
//
