LOCUS       sequence-263469-        7280 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 7280)
  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 7280)
  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: pNIC28-Bsa4
FEATURES             Location/Qualifiers
     source          1..7280
                     /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             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     promoter        170..615
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(616..702,703..2037)
                     /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             2145..2162
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2215..2233)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2229..2276
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2313..2768
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2400..2419)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2610..2631
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2860..3675)
                     /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(2860..3675)
                     /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     3583..3602
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3667..3686
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3797..4385
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4286..4305
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4539..4556
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4571..4710
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4636..4655)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4796..4818
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4812..5003)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5812..6894)
                     /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     6856..6875
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6895..6972)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7178..7197
                     /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 agatctgggt accgagaacc tgtacttcca atccatggag accgacgtcc acatatacct
      181 gccgttcact attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa
      241 aggcaacttt atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag
      301 atagattttt tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa
      361 caaaagagga aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa
      421 agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca
      481 aagtgtatac tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact
      541 aacttgccat cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa
      601 aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt
      661 actaccgcac tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag
      721 ccatataagg aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct
      781 gaacagcaaa aaaatgaaaa atataaagtt cctgagttcg attcgtccac aattaaaaat
      841 atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cactgacggc
      901 actgtcgcaa actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat
      961 gcggatgaca catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc
     1021 tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc
     1081 ctaaaagacc aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc
     1141 cgtttattct acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca
     1201 caagttaacg tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa
     1261 tcaatctttg acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc
     1321 aactacagct caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc
     1381 cacaaatact tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa
     1441 tctttattta acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa
     1501 aaacttctgc aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg
     1561 attgagctaa acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac
     1621 acagtaacag atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg
     1681 ttcactgact cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac
     1741 atgcttggtt atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc
     1801 cttgtgttaa aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct
     1861 gtacctcaag cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc
     1921 tacgcagaca aacaatcaac gtttgcgcct agcttcctgc tgaacatcaa aggcaagaaa
     1981 acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataaaaa
     2041 cgcaaaagaa aatgccgata tcctattggc attgacggtc tccagtaaag gtggatacgg
     2101 atccgaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
     2161 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     2221 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     2281 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     2341 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2401 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2461 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2521 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2581 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2641 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2701 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     2761 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2821 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     2881 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     2941 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     3001 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     3061 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     3121 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     3181 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     3241 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     3301 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     3361 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     3421 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     3481 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     3541 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     3601 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     3661 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     3721 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3781 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3841 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3901 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3961 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4021 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4081 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4141 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4201 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4261 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4321 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4381 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4441 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4501 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4561 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4621 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     4681 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     4741 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4801 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     4861 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     4921 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     4981 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     5041 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     5101 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     5161 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     5221 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     5281 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     5341 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     5401 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     5461 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     5521 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     5581 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     5641 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     5701 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     5761 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     5821 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5881 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     5941 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     6001 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     6061 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     6121 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     6181 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     6241 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     6301 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     6361 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     6421 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     6481 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     6541 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     6601 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     6661 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     6721 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     6781 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     6841 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     6901 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     6961 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     7021 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     7081 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     7141 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     7201 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     7261 agatctcgat cccgcgaaat
//
