LOCUS       sequence-12257-e        7260 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 7260)
  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 7260)
  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-CTHF
FEATURES             Location/Qualifiers
     source          1..7260
                     /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)."
     promoter        96..528
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(529..615,616..1950)
                     /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             2009..2029
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2030..2047
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2048..2071
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2121..2138
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2191..2209)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2205..2252
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2289..2744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2376..2395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2586..2607
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2837..3652)
                     /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(2837..3652)
                     /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     3560..3579
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3644..3663
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3774..4362
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4263..4282
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4516..4533
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4548..4690
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4616..4635)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4776..4798
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4792..4983)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5792..6874)
                     /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     6836..6855
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6875..6952)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7158..7177
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaacct taagaaggag atatactatg caggtcgttc actattattt agtgaaatga
      121 gatattatga tattttctga attgtgatta aaaaggcaac tttatgccca tgcaacagaa
      181 actataaaaa atacagagaa tgaaaagaaa cagatagatt ttttagttct ttaggcccgt
      241 agtctgcaaa tccttttatg attttctatc aaacaaaaga ggaaaataga ccagttgcaa
      301 tccaaacgag agtctaatag aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat
      361 aaagcaggca agacctaaaa tgtgtaaagg gcaaagtgta tactttggcg tcacccctta
      421 catattttag gtcttttttt attgtgcgta actaacttgc catcttcaaa caggagggct
      481 ggaagaagca gaccgctaac acagtacata aaaaaggaga catgaacgat gaacatcaaa
      541 aagtttgcaa aacaagcaac agtattaacc tttactaccg cactgctggc aggaggcgca
      601 actcaagcgt ttgcgaaaga aacgaaccaa aagccatata aggaaacata cggcatttcc
      661 catattacac gccatgatat gctgcaaatc cctgaacagc aaaaaaatga aaaatataaa
      721 gttcctgagt tcgattcgtc cacaattaaa aatatctctt ctgcaaaagg cctggacgtt
      781 tgggacagct ggccattaca aaacactgac ggcactgtcg caaactatca cggctaccac
      841 atcgtctttg cattagccgg agatcctaaa aatgcggatg acacatcgat ttacatgttc
      901 tatcaaaaag tcggcgaaac ttctattgac agctggaaaa acgctggccg cgtctttaaa
      961 gacagcgaca aattcgatgc aaatgattct atcctaaaag accaaacaca agaatggtca
     1021 ggttcagcca catttacatc tgacggaaaa atccgtttat tctacactga tttctccggt
     1081 aaacattacg gcaaacaaac actgacaact gcacaagtta acgtatcagc atcagacagc
     1141 tctttgaaca tcaacggtgt agaggattat aaatcaatct ttgacggtga cggaaaaacg
     1201 tatcaaaatg tacagcagtt catcgatgaa ggcaactaca gctcaggcga caaccatacg
     1261 ctgagagatc ctcactacgt agaagataaa ggccacaaat acttagtatt tgaagcaaac
     1321 actggaactg aagatggcta ccaaggcgaa gaatctttat ttaacaaagc atactatggc
     1381 aaaagcacat cattcttccg tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc
     1441 acggctgagt tagcaaacgg cgctctcggt atgattgagc taaacgatga ttacacactg
     1501 aaaaaagtga tgaaaccgct gattgcatct aacacagtaa cagatgaaat tgaacgcgcg
     1561 aacgtcttta aaatgaacgg caaatggtac ctgttcactg actcccgcgg atcaaaaatg
     1621 acgattgacg gcattacgtc taacgatatt tacatgcttg gttatgtttc taattcttta
     1681 actggcccat acaagccgct gaacaaaact ggccttgtgt taaaaatgga tcttgatcct
     1741 aacgatgtaa cctttactta ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc
     1801 gtgattacaa gctatatgac aaacagagga ttctacgcag acaaacaatc aacgtttgcg
     1861 cctagcttcc tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt
     1921 gaacaaggac aattaacagt taacaaataa aaacgcaaaa gaaaatgccg atatcctatt
     1981 ggcattgacg tcaggtggca cacctgcaga gaacctctac ttccaatcgc accatcatca
     2041 ccaccatgat tacaaggatg acgacgataa gtgaggatcc gaattcgagc tccgtcgaca
     2101 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     2161 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     2221 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     2281 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2341 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2401 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2461 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2521 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2581 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2641 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2701 aaaatttaac gcgaatttta acaaaatatt aacgtttaca atttcaggtg gcacttttcg
     2761 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2821 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2881 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2941 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     3001 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     3061 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     3121 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     3181 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     3241 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     3301 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     3361 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3421 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3481 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3541 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3601 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3661 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     3721 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3781 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3841 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3901 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3961 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4021 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4081 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4141 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4201 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4261 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4321 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4381 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4441 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4501 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4561 tttctcctta cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa
     4621 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4681 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4741 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4801 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     4861 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     4921 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     4981 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     5041 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     5101 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     5161 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     5221 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     5281 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     5341 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     5401 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     5461 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     5521 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     5581 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     5641 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     5701 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     5761 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5821 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5881 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     5941 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     6001 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     6061 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     6121 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     6181 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     6241 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     6301 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     6361 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     6421 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     6481 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     6541 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     6601 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     6661 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     6721 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     6781 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     6841 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     6901 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     6961 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     7021 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     7081 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     7141 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     7201 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
