LOCUS       sequence-434035-        5844 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  heterologous expression of PA1472 for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5844)
  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 5844)
  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_PA1472_Nterm_His
FEATURES             Location/Qualifiers
     source          1..5844
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     141..160
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     244..263
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        244..262
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    263..287
                     /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             302..324
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             343..360
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             370..387
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             952..969
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1022..1040)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1036..1083
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1120..1575
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1207..1226)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1417..1438
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1667..2482)
                     /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(1667..2482)
                     /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     2390..2409
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2474..2493
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2604..3192
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3093..3112
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3346..3363
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3378..3517
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3443..3462)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3603..3625
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3619..3810)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4619..5701)
                     /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     5663..5682
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5702..5779)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
       61 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
      121 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
      181 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
      241 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
      301 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac
      361 agcagcggcc tggtgccgcg cggcagccat atgaactttg tttttccgca tccgctggat
      421 acaccgcgtc tgcgtctgcg cacctttcgt gaagaagatg cagcaccgct gtttgcaatg
      481 atgagcgatc cggaagttat gcgttattgg aatacccctc cgtggaccac accggcacag
      541 gcacgtgaag caattctgcg tgatagccag gcactggttg atggtgaata tctgaatctg
      601 gcaattgaac gtcgtgaaga tggtcagctg ctgggtagct gtattctgtt tcattttgaa
      661 aaaggtagcc gtcgtgcaga actgggttat tgtctggcac gtgcagcaca aggtcgtggt
      721 tatatgggtg aagcactgcg tcgtctgctg gcatttgcat ttgatgaaat tgatctgaat
      781 cgtctggaag cagaaatcga tccgcgtaat cgtccgagcg cagcaagcct ggaacgtctg
      841 ggttttcgtc aagaaggcct gctggcacag cgttggattg ttagcggtga agttagcgat
      901 agcgcactgt atggactgct ggcagaacat tggcgtaatc gctaactcga gcaccaccac
      961 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1021 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1081 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1141 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1201 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1261 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1321 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1381 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1441 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1501 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1561 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1621 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     1681 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     1741 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     1801 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     1861 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     1921 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     1981 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     2041 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     2101 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     2161 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     2221 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     2281 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     2341 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     2401 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     2461 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     2521 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2581 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2641 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2701 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2761 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2821 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2881 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2941 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3001 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3061 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3121 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3181 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3241 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3301 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3361 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3421 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3481 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3541 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3601 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3661 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3721 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     3781 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     3841 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     3901 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     3961 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     4021 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     4081 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     4141 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     4201 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     4261 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     4321 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     4381 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     4441 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     4501 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     4561 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     4621 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4681 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     4741 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     4801 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     4861 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     4921 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     4981 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     5041 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     5101 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     5161 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     5221 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     5281 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     5341 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     5401 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     5461 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     5521 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     5581 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     5641 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     5701 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     5761 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     5821 aagcagccca gtagtaggtt gagg
//
