LOCUS       sequence-239406-        7598 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 7598)
  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 7598)
  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: pNH-TrxT
FEATURES             Location/Qualifiers
     source          1..7598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             45..62
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             72..398
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             405..425
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     promoter        441..886
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(887..973,974..2308)
                     /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             2416..2433
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2486..2504)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2500..2547
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2584..3039
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2671..2690)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2881..2902
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3131..3946)
                     /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(3131..3946)
                     /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     3854..3873
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3938..3957
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4068..4656
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4557..4576
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4810..4827
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4842..4981
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4907..4926)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5067..5089
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5083..5274)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6083..7165)
                     /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     7127..7146
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7166..7243)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7449..7468
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7552..7571
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7552..7570
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7571..7595
                     /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)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc
       61 attcttctgg tatgagcgat aaaattattc acctgactga cgacagtttt gacacggatg
      121 tactcaaagc ggacggggcg atcctcgtcg atttctgggc agagtggtgc ggtccgtgca
      181 aaatgatcgc cccgattctg gatgaaatcg ctgacgaata tcagggcaaa ctgaccgttg
      241 caaaactgaa catcgatcaa aaccctggca ctgcgccgaa atatggcatc cgtggtatcc
      301 cgactctgct gctgttcaaa aacggtgaag tggcggcaac caaagtgggc gcactgtcta
      361 aaggtcagtt gaaagagttc ctcgacgcta acctggccgg taccgagaac ttgtacttcc
      421 aatccatgga gaccgacgtc cacatatacc tgccgttcac tattatttag tgaaatgaga
      481 tattatgata ttttctgaat tgtgattaaa aaggcaactt tatgcccatg caacagaaac
      541 tataaaaaat acagagaatg aaaagaaaca gatagatttt ttagttcttt aggcccgtag
      601 tctgcaaatc cttttatgat tttctatcaa acaaaagagg aaaatagacc agttgcaatc
      661 caaacgagag tctaatagaa tgaggtcgaa aagtaaatcg cgcgggtttg ttactgataa
      721 agcaggcaag acctaaaatg tgtaaagggc aaagtgtata ctttggcgtc accccttaca
      781 tattttaggt ctttttttat tgtgcgtaac taacttgcca tcttcaaaca ggagggctgg
      841 aagaagcaga ccgctaacac agtacataaa aaaggagaca tgaacgatga acatcaaaaa
      901 gtttgcaaaa caagcaacag tattaacctt tactaccgca ctgctggcag gaggcgcaac
      961 tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag gaaacatacg gcatttccca
     1021 tattacacgc catgatatgc tgcaaatccc tgaacagcaa aaaaatgaaa aatataaagt
     1081 tcctgagttc gattcgtcca caattaaaaa tatctcttct gcaaaaggcc tggacgtttg
     1141 ggacagctgg ccattacaaa acactgacgg cactgtcgca aactatcacg gctaccacat
     1201 cgtctttgca ttagccggag atcctaaaaa tgcggatgac acatcgattt acatgttcta
     1261 tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac gctggccgcg tctttaaaga
     1321 cagcgacaaa ttcgatgcaa atgattctat cctaaaagac caaacacaag aatggtcagg
     1381 ttcagccaca tttacatctg acggaaaaat ccgtttattc tacactgatt tctccggtaa
     1441 acattacggc aaacaaacac tgacaactgc acaagttaac gtatcagcat cagacagctc
     1501 tttgaacatc aacggtgtag aggattataa atcaatcttt gacggtgacg gaaaaacgta
     1561 tcaaaatgta cagcagttca tcgatgaagg caactacagc tcaggcgaca accatacgct
     1621 gagagatcct cactacgtag aagataaagg ccacaaatac ttagtatttg aagcaaacac
     1681 tggaactgaa gatggctacc aaggcgaaga atctttattt aacaaagcat actatggcaa
     1741 aagcacatca ttcttccgtc aagaaagtca aaaacttctg caaagcgata aaaaacgcac
     1801 ggctgagtta gcaaacggcg ctctcggtat gattgagcta aacgatgatt acacactgaa
     1861 aaaagtgatg aaaccgctga ttgcatctaa cacagtaaca gatgaaattg aacgcgcgaa
     1921 cgtctttaaa atgaacggca aatggtacct gttcactgac tcccgcggat caaaaatgac
     1981 gattgacggc attacgtcta acgatattta catgcttggt tatgtttcta attctttaac
     2041 tggcccatac aagccgctga acaaaactgg ccttgtgtta aaaatggatc ttgatcctaa
     2101 cgatgtaacc tttacttact cacacttcgc tgtacctcaa gcgaaaggaa acaatgtcgt
     2161 gattacaagc tatatgacaa acagaggatt ctacgcagac aaacaatcaa cgtttgcgcc
     2221 tagcttcctg ctgaacatca aaggcaagaa aacatctgtt gtcaaagaca gcatccttga
     2281 acaaggacaa ttaacagtta acaaataaaa acgcaaaaga aaatgccgat atcctattgg
     2341 cattgacggt ctccagtaaa ggtggatacg gatccgaatt cgagctccgt cgacaagctt
     2401 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     2461 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     2521 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     2581 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2641 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2701 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2761 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2821 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2881 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2941 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3001 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     3061 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3121 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     3181 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     3241 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     3301 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     3361 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     3421 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     3481 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     3541 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     3601 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     3661 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     3721 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     3781 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     3841 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     3901 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     3961 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     4021 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4081 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4141 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4201 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4261 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4321 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4381 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4441 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4501 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4561 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4621 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4681 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4741 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4801 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4861 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     4921 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4981 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5041 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5101 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     5161 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     5221 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     5281 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     5341 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     5401 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     5461 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     5521 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     5581 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     5641 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     5701 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     5761 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     5821 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     5881 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     5941 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     6001 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     6061 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6121 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     6181 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     6241 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     6301 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     6361 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     6421 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     6481 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     6541 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     6601 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     6661 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     6721 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     6781 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     6841 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     6901 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     6961 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     7021 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     7081 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     7141 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     7201 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     7261 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     7321 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     7381 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     7441 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     7501 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
     7561 tcactatagg ggaattgtga gcggataaca attcccct
//
