LOCUS       sequence-437239-        5784 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 5784)
  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 5784)
  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_Cterm_His
FEATURES             Location/Qualifiers
     source          1..5784
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..71
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(65..256)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1065..2147)
                     /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     2109..2128
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2148..2225)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2431..2450
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2534..2553
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2534..2552
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2553..2577
                     /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             2592..2614
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3182..3199
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3252..3270)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3266..3313
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3350..3805
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3437..3456)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3647..3668
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3897..4712)
                     /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(3897..4712)
                     /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     4620..4639
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4704..4723
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4834..5422
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5323..5342
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5576..5593
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5608..5747
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5673..5692)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
       61 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
      121 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
      181 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
      241 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
      301 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
      361 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
      421 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
      481 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
      541 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
      601 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
      661 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
      721 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
      781 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
      841 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
      901 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
      961 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     1021 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     1081 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1141 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     1201 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     1261 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     1321 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     1381 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     1441 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     1501 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     1561 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     1621 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     1681 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     1741 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     1801 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     1861 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     1921 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     1981 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     2041 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     2101 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     2161 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     2221 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     2281 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     2341 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     2401 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     2461 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     2521 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     2581 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgatgaact ttgtttttcc
     2641 gcatccgctg gatacaccgc gtctgcgtct gcgcaccttt cgtgaagaag atgcagcacc
     2701 gctgtttgca atgatgagcg atccggaagt tatgcgttat tggaataccc ctccgtggac
     2761 cacaccggca caggcacgtg aagcaattct gcgtgatagc caggcactgg ttgatggtga
     2821 atatctgaat ctggcaattg aacgtcgtga agatggtcag ctgctgggta gctgtattct
     2881 gtttcatttt gaaaaaggta gccgtcgtgc agaactgggt tattgtctgg cacgtgcagc
     2941 acaaggtcgt ggttatatgg gtgaagcact gcgtcgtctg ctggcatttg catttgatga
     3001 aattgatctg aatcgtctgg aagcagaaat cgatccgcgt aatcgtccga gcgcagcaag
     3061 cctggaacgt ctgggttttc gtcaagaagg cctgctggca cagcgttgga ttgttagcgg
     3121 tgaagttagc gatagcgcac tgtatggact gctggcagaa cattggcgta atcgcctcga
     3181 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     3241 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     3301 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     3361 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3421 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3481 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3541 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3601 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3661 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3721 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3781 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3841 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
     3901 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     3961 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     4021 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     4081 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     4141 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     4201 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     4261 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     4321 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     4381 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     4441 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     4501 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     4561 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     4621 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     4681 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     4741 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4801 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4861 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4921 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4981 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5041 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5101 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5161 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5221 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5281 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5341 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5401 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5461 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5521 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     5581 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     5641 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5701 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     5761 caacacccgc tgacgcgccc tgac
//
