LOCUS       sequence-437238-        5736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  heterologous expression of PA4114 for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5736)
  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 5736)
  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_PA4114_Cterm_His
FEATURES             Location/Qualifiers
     source          1..5736
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..52
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(46..237)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1046..2128)
                     /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     2090..2109
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2129..2206)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2412..2431
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2515..2534
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2515..2533
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2534..2558
                     /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             2573..2595
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3115..3132
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3185..3203)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3199..3246
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3283..3738
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3370..3389)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3580..3601
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3830..4645)
                     /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(3830..4645)
                     /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     4553..4572
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4637..4656
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4767..5355
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5256..5275
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5509..5526
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5541..5680
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5606..5625)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
       61 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
      121 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
      181 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
      241 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
      301 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
      361 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
      421 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
      481 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
      541 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
      601 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
      661 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
      721 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
      781 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
      841 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
      901 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
      961 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     1021 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     1081 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     1141 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     1201 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     1261 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     1321 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     1381 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     1441 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     1501 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     1561 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     1621 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     1681 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     1741 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     1801 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     1861 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     1921 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     1981 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     2041 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     2101 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     2161 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     2221 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     2281 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     2341 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     2401 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     2461 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     2521 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     2581 actttaagaa ggagatatac catgattgaa ctgcgtacca ttacacgtga tgattgggaa
     2641 acctgtattg atctgaaagt tgcacgtcat caggcacatt ttgttgcaag caatctgtat
     2701 agcctggcac agagccgttt tctgcctggt tttcgtgcag ttggtattca tcatggtggt
     2761 cgtatggttg gttttgcact gtatggtccg gatgcagatg atggtcatta ttggctgtat
     2821 cgtctgatga ttgatcgtcg ctatcaaggt aaaggtcatg gtcgtgcagc actgcagcgt
     2881 attattgcaa gcgttcgtcg tacccgtcgt cgtagcgatc gcctgatggt tgcatatgcc
     2941 gttgataatg atgttgcccg tcagctgtac cgtgatgcag gttttgttga acaaggtctg
     3001 gcaccgtggg gtgaaaccat ggcatgtctg atgctgccgg aaccgagcca tcgtagcggt
     3061 aaagttgccg gtgcaccagg tggtcgcgca gcccatggtg ttccggcact cgagcaccac
     3121 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     3181 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     3241 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     3301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     3721 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3781 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     3841 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     3901 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     3961 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     4021 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     4081 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     4141 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     4201 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     4261 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     4321 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     4381 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     4441 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     4501 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     4561 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     4621 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     4681 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4741 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4801 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4861 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4921 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4981 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5041 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5101 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5161 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5221 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5281 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5341 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5401 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5461 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     5521 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5581 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     5641 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     5701 cgctgacgcg ccctgacggg cttgtctgct cccggc
//
