LOCUS       sequence-434036-        5823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  heterologous expression of PA1377 for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5823)
  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 5823)
  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_PA1377_Nterm_His
FEATURES             Location/Qualifiers
     source          1..5823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..44
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(64..141)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     347..366
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     450..469
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        450..468
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    469..493
                     /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             508..530
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             549..566
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             576..593
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1137..1154
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1207..1225)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1221..1268
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1305..1760
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1392..1411)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1602..1623
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1852..2667)
                     /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(1852..2667)
                     /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     2575..2594
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2659..2678
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2789..3377
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3278..3297
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3531..3548
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3563..3702
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3628..3647)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3788..3810
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3804..3995)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(4804..5823,1..63))
                     /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"
ORIGIN
        1 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
       61 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
      121 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
      181 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
      241 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
      301 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
      361 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
      421 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
      481 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
      541 gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatga
      601 ccgcagaaag cccgaccatt cgtctggaac gttatagcga acgtcatgtt gaaggtctga
      661 ccgcactgta taatgatccg gcagttgcac gtcaggttct gcagatgccg tatcagagcg
      721 ttgaacagcg tcgtaaacgt ctgcatgata gcgcagatga tgatcgtctg ctgattctgg
      781 ttgcactgca tcagggtgat gttattggta gcgcaagcct ggaacagcat ccgcgtattc
      841 gtcgtagcca tagcggtagc attggtatgg gtgttgcagt tgcatggcaa ggtaaaggtg
      901 ttggtagccg tctgctgggt gaactgctgg atattgcaga taattggatg aatctgcgtc
      961 gtgttgaact gaccgtttat accgataatg caccggcact ggcactgtat cgtaaatttg
     1021 gttttgaaac cgaaggtgag atgcgtgatt atgcagttcg tgatggtcgt tttgttgatg
     1081 tttatagcat ggcacgtctg cgtcgcgtgg aaggtcgtgt tggtgaataa ctcgagcacc
     1141 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1201 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1261 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1321 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1381 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1441 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1501 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1561 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1621 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1681 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1741 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1801 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     1861 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     1921 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     1981 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     2041 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     2101 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2161 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2221 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2281 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2341 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2401 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     2461 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     2521 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     2581 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     2641 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     2701 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2761 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2821 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2881 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2941 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3001 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3061 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3121 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3181 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3241 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3301 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3361 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3421 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3481 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3541 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3601 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3661 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3721 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3781 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3841 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3901 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3961 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     4021 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     4081 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     4141 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     4201 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4261 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4321 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4381 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4441 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     4501 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4561 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     4621 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     4681 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4741 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4801 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4861 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     4921 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     4981 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     5041 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     5101 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5161 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5221 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5281 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5341 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5401 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5461 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5521 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5581 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5641 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5701 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5761 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5821 gcg
//
