LOCUS       sequence-321834-        6652 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Exression of ATI QconCAT protein in E. coli expression strains 
            (BL21). Encodes a concatemer of tryptic peptides from wheat 
            amylase/trypsin inhibitors. Used as standard for LC-MS-based 
            quantification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6652)
  AUTHORS   Sielaff M, Curella V, Neerukonda M, Afzal M, El Hassouni K, Distler 
            U, Schuppan D, Longin CFH, Tenzer S
  TITLE     Hybrid QconCAT-Based Targeted Absolute and Data-Independent 
            Acquisition-Based Label-Free Quantification Enables In-Depth 
            Proteomic Characterization of Wheat Amylase/Trypsin Inhibitor 
            Extracts.
  JOURNAL   J Proteome Res. 2021 Jan 28. doi: 10.1021/acs.jproteome.0c00752.
  PUBMED    33507751
REFERENCE   2  (bases 1 to 6652)
  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: "J Proteome 
            Res. 2021 Jan 28. doi: 10.1021/acs.jproteome.0c00752."
COMMENT     Sequence Label: pET26b(+)-ATI_QconCAT_1
FEATURES             Location/Qualifiers
     source          1..6652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1517..1534
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1587..1605)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1601..1648
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1685..2140
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1772..1791)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1982..2003
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2232..3047)
                     /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(2232..3047)
                     /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     2955..2974
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3039..3058
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3169..3757
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3658..3677
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3911..3928
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3943..4082
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4008..4027)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4168..4190
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4184..4375)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5184..6266)
                     /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     6228..6247
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6267..6344)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6550..6569
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat ggcgggcaaa tacgttgttg ataccagcaa
      121 actgaccgcg gcgagcatta ccgcggtgtg ccgtctgccg attgttgttg atgcgagcgg
      181 tgacggcgcg tacgtgtgca aagattgctg ccagcaactg gcggacatca gcgagtggtg
      241 ccgtagccac aacagcggtc cgtggagctg gtgcaacccg gcgaccggct ataagctgca
      301 gtgcgttggt agccaagtgc cggaggcggt tctgcgtgac tgctgccaac aactggcgga
      361 catcaacaac gaatggtgcc gtagcgttta ccaggagctg ggtgtgcgtg aatatgttgc
      421 gcagcaaacc tgcggtgtgg gcattgttgg cagcccggtg agcaccgagc cgggtaacac
      481 cccgcgtgat tacgttctgc agcaaacctg cggcaccttc accccgggta gcaaactgcc
      541 ggaatggatg accagcgcga gcatctttag cccgatgaag ccgtacctgg cgaaactgta
      601 ttgctgccag gagctggcgg aaattccgca gcaatgccgt ctgaccgcgg cgagcatcac
      661 cgcggtgtgc aagctgccga tcgttattga cgcgagcggt ggccgttact ttattgcgct
      721 gccggtgccg agccaaccgg ttgatccgcg tagcggtaac gtgggcgaga gcggtctgat
      781 tgacctgccg ggttgcccgc gtgaatatgt ggcgcagcaa acctgcggca tcagcattag
      841 cggtagcgcg gttagcaccg aaccgggcaa caccccgcgt gattgctgcc aacagctggc
      901 ggacatcagc gaatggccgc gtatggcggt taccctgtgg agcatttggt atgcgcgtag
      961 ccacaatagc ggcccgtgga gctggtgcga tccggcgacc ggttacaagc tgtactgctg
     1021 ccaagagctg gcggaaatca gccagcaatg ccgtattgaa accccgggca gcccgtacct
     1081 ggcgaaacag caatgctgcg gcgagctggc gaacatcccg caacagtgcc gtattgaaat
     1141 gccgggtccg ccgtatctgg cgaagcagga gtgctgcgaa caactggcga acatcccgca
     1201 gcaatgccgt accagcattt acacctgcta tgagggtgtg ggcctgccgg ttgatccgct
     1261 gcagggttgc cactactatg tgaccagcca aacctgcggt tttgtgccgc tgctgccgat
     1321 cgaagttatg aaattaactg cggcgagcgt gccggcggtt tgcaaggtgc cggcgctgcc
     1381 gggttgccgt ccggttctga aactgcagtg cgtgggtaac caagttccgg aagcgattca
     1441 gcgtgactgc tgccaacaac tggcgcatat cagcgaatgg tgccgcatcg gtgactacgc
     1501 gggtatcaag ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     1561 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1621 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     1681 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1741 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1801 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1861 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1921 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1981 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2041 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2101 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     2161 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2221 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     2281 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     2341 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     2401 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     2461 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     2521 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2581 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2641 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2701 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     2761 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2821 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     2881 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     2941 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     3001 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     3061 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     3121 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3181 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3241 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3301 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3361 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3421 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3481 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3541 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3601 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3661 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3721 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3781 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3841 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3901 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3961 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     4021 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     4081 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4141 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4201 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     4261 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     4321 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     4381 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     4441 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4501 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4561 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4621 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4681 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4741 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4801 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4861 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4921 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4981 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     5041 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     5101 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     5161 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5221 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     5281 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     5341 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     5401 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     5461 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5521 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5581 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5641 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5701 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5761 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5821 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5881 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5941 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     6001 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     6061 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     6121 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     6181 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     6241 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     6301 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     6361 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     6421 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6481 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6541 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     6601 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa at
//
