LOCUS       sequence-434034-        5850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  heterologous expression for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5850)
  AUTHORS   Mayers JR, Varon J, Zhou RR, Daniel-Ivad M, Beaulieu C, Bholse A, 
            Glasser NR, Lichtenauer FM, Ng J, Vera MP, Huttenhower C, Perrella 
            MA, Clish CB, Zhao SD, Baron RM, Balskus EP
  TITLE     A metabolomics pipeline highlights microbial metabolism in 
            bloodstream infections.
  JOURNAL   Cell. 2024 Jun 11:S0092-8674(24)00579-8. doi: 
            10.1016/j.cell.2024.05.035.
  PUBMED    38885650
REFERENCE   2  (bases 1 to 5850)
  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: "Cell. 2024 
            Jun 11:S0092-8674(24)00579-8. doi: 10.1016/j.cell.2024.05.035."
COMMENT     Sequence Label: pET28a_speG_Nterm_His
FEATURES             Location/Qualifiers
     source          1..5850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     67..89
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(83..274)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1083..2165)
                     /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     2127..2146
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2166..2243)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2449..2468
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2552..2571
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2552..2570
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2571..2595
                     /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             2610..2632
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2651..2668
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2678..2695
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3266..3283
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3336..3354)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3350..3397
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3434..3889
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3521..3540)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3731..3752
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3981..4796)
                     /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(3981..4796)
                     /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     4704..4723
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4788..4807
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4918..5506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5407..5426
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5660..5677
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5692..5831
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5757..5776)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
       61 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
      121 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
      181 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
      241 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
      301 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
      361 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
      421 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
      481 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
      541 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
      601 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
      661 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
      721 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
      781 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
      841 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
      901 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
      961 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     1021 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     1081 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     1141 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     1201 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     1261 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     1321 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     1381 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     1441 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     1501 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     1561 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     1621 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     1681 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     1741 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     1801 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     1861 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     1921 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     1981 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     2041 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     2101 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     2161 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     2221 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     2281 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     2341 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     2401 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     2461 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     2521 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     2581 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat
     2641 gggcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat
     2701 gccaagcgcc cacagtgtta agctacgccc gctggagcgt gaagatttac gctatgtaca
     2761 tcaactcgac aataacgcca gtgtgatgcg ttactggttt gaggaaccct acgaagcctt
     2821 tgttgaactc tctgatctgt atgataagca tattcacgat cagagcgaac ggcgctttgt
     2881 ggtggaatgt gacggcgaaa aagccggtct ggtggagctg gtggaaatta accatgttca
     2941 tcgccgcgca gaatttcaga taattatctc cccggagtat caggggaaag gtctggcaac
     3001 ccgtgccgcc aaattagcaa tggactatgg ctttaccgtt ctcaatctct ataagctgta
     3061 tctgatcgtt gataaagaga atgaaaaagc gattcacatt taccgcaagc ttggcttttc
     3121 ggttgaaggt gaattgatgc acgagttctt tattaatggt caatatcgta atgccattcg
     3181 catgtgtata ttccagcatc agtatctggc agagcacaaa acaccgggtc agactctcct
     3241 gaagccgacc gcacaatagc tcgagcacca ccaccaccac cactgagatc cggctgctaa
     3301 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     3361 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     3421 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3481 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3541 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3601 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3661 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3721 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3781 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3841 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     3901 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3961 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     4021 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     4081 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     4141 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     4201 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     4261 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     4321 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     4381 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     4441 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     4501 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     4561 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     4621 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     4681 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     4741 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     4801 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     4861 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4921 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4981 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5041 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     5101 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5161 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5221 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5281 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5341 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5401 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5461 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5521 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5581 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     5641 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     5701 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     5761 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     5821 tcatggctgc gccccgacac ccgccaacac
//
