LOCUS       sequence-126545-        6167 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  construct for expression of OmpT protease in the pET28a plasmid 
            using the restriction sites NcoI and EcoRI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6167)
  AUTHORS   Wu C, Tran JC, Zamdborg L, Durbin KR, Li M, Ahlf DR, Early BP, 
            Thomas PM, Sweedler JV, Kelleher NL
  TITLE     A protease for 'middle-down' proteomics.
  JOURNAL   Nat Methods. 2012 Jun 17;9(8):822-4. doi: 10.1038/nmeth.2074.
  PUBMED    22706673
REFERENCE   2  (bases 1 to 6167)
  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"; doi: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2012-06-17- 17"; volume: "9"; 
            issue: "8"; pages: "822-4"
COMMENT     Sequence Label: pET28-OmpT
FEATURES             Location/Qualifiers
     source          1..6167
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1104..1126
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1141..1165
                     /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)."
     primer_bind     complement(1165..1184)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1166..1184)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1268..1287)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1493..1570
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1571..2653
                     /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     complement(1590..1609)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3462..3653
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3647..3669)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3755..3897
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3810..3829
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3912..3929)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4083..4671)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4163..4182)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4782..4801)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4793..5608
                     /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             4793..5608
                     /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     complement(4866..4885)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5701..6156)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5838..5859)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6050..6069
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattctta aaatgtgtac ttaagaccag cagtagtgat gaagttatag
      241 ttttctatac ctgctccatt tttgctgtag tctgaagtgt tattattgtg atcataaagt
      301 gaagtattac cttttttatt cgtaacccga ttccatgcgc cttcaacata aacttttgcg
      361 ttaggtgtga cgtaataacc tgcattgact gcaacagaat agtaattttg gtctttgacc
      421 ttactgcgat aagtgattct ttttcccggg tcatagtgtt catcgttatc agatgattcc
      481 acccagccgc tgtatttaaa tgtgccaccg agttcaaaat cttcataacg ataacttcca
      541 gtcaagccaa tgtagggcat tttaaaacgt tgtttgtagc cgattgctct ttctccattc
      601 gggaaggagc cgatatcatc tctgaatccc tcctcagaac tgtagatata ggaaccacct
      661 ctggctgtaa agctataacg gctttcctga tatccggcca tgagtcccag gcggtaattg
      721 ggttcgttga ggagccagcc tttgatattc agatcaaatt cgttggcata attgagttgt
      781 gtatcagggt gtctactttc atccgtccag gttccggggt tactggaatc catccagtcc
      841 tgatcgacca tattgccacc tcggctgccg agagttgtcc agccagcagc cccgatagat
      901 atctggggca tcaaatccca attaattgca cctttaataa ttgcagcgtt attgaatttc
      961 cagtcgagtt gactgacttt tcggcctcct tcttcggcta gataaacacg ctcttttgtt
     1021 tttccgctca gagttccaag actaatgtcc gcatttatgt tgtcaggagt aaacgataaa
     1081 gtctcggtag aagccatggt atatctcctt cttaaagtta aacaaaatta tttctagagg
     1141 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
     1201 atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
     1261 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
     1321 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
     1381 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
     1441 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat
     1501 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     1561 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     1621 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     1681 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     1741 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     1801 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     1861 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     1921 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     1981 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     2041 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     2101 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     2161 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     2221 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     2281 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     2341 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     2401 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     2461 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     2521 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     2581 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2641 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat
     2701 ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     2761 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     2821 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     2881 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     2941 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg
     3001 tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg
     3061 gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt
     3121 ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa
     3181 cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     3241 taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt
     3301 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc
     3361 gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca
     3421 ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa
     3481 ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
     3541 acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg
     3601 agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     3661 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     3721 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     3781 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     3841 ccatatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     3901 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3961 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4021 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4081 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4141 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4201 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4261 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4321 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4381 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4441 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4501 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     4561 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4621 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4681 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4741 gtcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     4801 tattcaacgg gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt
     4861 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt
     4921 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     4981 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     5041 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     5101 gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     5161 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     5221 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     5281 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     5341 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     5401 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     5461 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     5521 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     5581 tcatttgatg ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat
     5641 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5701 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     5761 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     5821 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     5881 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     5941 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     6001 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     6061 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     6121 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
//
