LOCUS       sequence-394912-        7705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant Triticum
            aestivum Metallothionein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7705)
  AUTHORS   Mosna K, Jurczak K, Krezel A
  TITLE     Differentiated Zn(II) binding affinities in animal, plant, and 
            bacterial metallothioneins define their zinc buffering capacity at 
            physiological pZn.
  JOURNAL   Metallomics. 2023 Oct 4;15(10):mfad061. doi: 10.1093/mtomcs/mfad061.
  PUBMED    37804185
REFERENCE   2  (bases 1 to 7705)
  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: 
            "Metallomics."; date: "2023-10-4"; volume: "15(10):mfad061. doi"; 
            pages: " 10.1093/mtomcs/mfad061"
COMMENT     Sequence Label: Ec-1 Triticum aestivum Metallothionein
FEATURES             Location/Qualifiers
     source          1..7705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..110
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(111..179,180..971)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(329..348)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1013..1526)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1208..1229)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1420..1439
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1637..2225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2126..2145
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2379..2396
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2418..2437)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2578..2600
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2594..2785)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3342..4424)
                     /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     4386..4405
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4425..4502)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4655..4741
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4838..4924
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5021..5107
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5204..5290
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5387..5473
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5608..5627
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5608..5626
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5627..5651
                     /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             5666..5688
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5741..6559
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="5' region of the intein from the yeast Vma1 
                     subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 5' region
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
     CDS             6566..6718
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6722..7270
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="modified 3' region of the intein from the yeast 
                     Vma1 subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 3' region
                     /note="C-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="GHGGIRNNLNTENPLWDAIVGLGFLKDGVKNIPSFLSTDNIGTRE
                     TFLAGLIDSDGYVTDEHGIKATIKTIHTSVRDGLVSLARSLGLVVSVNAEPAKVDMNVT
                     KHKISYAIYMSGGDVLLNVLSKCAGSKKFRPAPAAAFARECRGFYFELQELKEDDYYGI
                     TLSDDSDHQFLLGSQVVVQN"
     primer_bind     complement(7590..7608)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7604..7651
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
       61 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
      121 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
      181 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
      241 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
      301 tcccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg
      361 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
      421 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
      481 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
      541 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
      601 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
      661 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
      721 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
      781 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
      841 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
      901 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
      961 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaccccggt
     1021 tgataatcag aaaagcccca aaaacaggaa gattgtataa gcaaatattt aaattgtaaa
     1081 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     1141 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga tagggttgag
     1201 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     1261 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt
     1321 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     1381 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     1441 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     1501 gcttaatgcg ccgctacagg gcgcgtaaaa ggatctaggt gaagatcctt tttgataatc
     1561 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1621 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1681 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1741 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1801 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1861 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1921 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1981 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2041 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2101 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2161 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2221 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2281 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2341 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2401 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     2461 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     2521 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     2581 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     2641 aaagctcatc agcgtggtcg tgcagcgatt cacagatgtc tgcctgttca tccgcgtcca
     2701 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     2761 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     2821 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     2881 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     2941 agagaaaaat cactcagggt caatgccagc cgaacgccag caagacgtag cccagcgcgt
     3001 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     3061 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     3121 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     3181 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     3241 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     3301 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     3361 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3421 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     3481 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     3541 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     3601 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
     3661 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     3721 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     3781 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     3841 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     3901 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     3961 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     4021 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     4081 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     4141 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     4201 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     4261 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     4321 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     4381 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     4441 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     4501 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     4561 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgccggcatg ccgccctttc
     4621 gtcttcaaga attaattccc aattccccag gcatcaaata aaacgaaagg ctcagtcgaa
     4681 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     4741 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     4801 cccgccataa actgccagga attaattccc caggcatcaa ataaaacgaa aggctcagtc
     4861 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     4921 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg ccsggagggk ggcgggcagg
     4981 acgcccgcca taaactgcca ggaattaatt ccccaggcat caaataaaac gaaaggctca
     5041 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
     5101 gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc
     5161 aggacgcccg ccataaactg ccaggaatta attccccagg catcaaataa aacgaaaggc
     5221 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     5281 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
     5341 ggcaggacgc ccgccataaa ctgccaggaa ttaattcccc aggcatcaaa taaaacgaaa
     5401 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     5461 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     5521 gcgggcagga cgcccgccat aaactgccag gaattgggga tcggaattaa ttcccggttt
     5581 aaaccgggga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     5641 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata tacttatgaa
     5701 aatcgaagaa ggtaaactgg taatcggttc cctggagggt tgctttgcca agggtaccaa
     5761 tgttttaatg gcggatgggt ctattgaatg tattgaaaac attgaggttg gtaataaggt
     5821 catgggtaaa gatggcagac ctcgtgaggt aattaaattg cccagaggaa gagaaactat
     5881 gtacagcgtc gtgcagaaaa gtcagcacag agcccacaaa agtgactcaa gtcgtgaagt
     5941 gccagaatta ctcaagttta cgtgtaatgc gacccatgag ttggttgtta gaacacctcg
     6001 tagtgtccgc cgtttgtctc gtaccattaa gggtgtcgaa tattttgaag ttattacttt
     6061 tgagatgggc caaaagaaag cccccgacgg tagaattgtt gagcttgtca aggaagtttc
     6121 aaagagctac ccaatatctg aggggcctga gagagccaac gaattagtag aatcctatag
     6181 aaaggcttca aataaagctt attttgagtg gactattgag gccagagatc tttctctgtt
     6241 gggttcccat gttcgtaaag ctacctacca gacttacgct ccaattcttt atgagaatga
     6301 ccactttttc gactacatgc aaaaaagtaa gtttcatctc accattgaag gtccaaaagt
     6361 acttgcttat ttacttggtt tatggattgg tgatggattg tctgacaggg caactttttc
     6421 ggttgattcc agagatactt ctttgatgga acgtgttact gaatatgctg aaaagttgaa
     6481 tttgtgcgcc gagtataagg acagaaaaga accacaagtt gccaaaactg ttaatttgta
     6541 ctctaaagtt gtcagaggtg ctagcacaaa tcctggtgta tccgcttggc aggtcaacac
     6601 agcttatact gcgggacaat tggtcacata taacggcaag acgtataaat gtttgcagcc
     6661 ccacacctcc ttggcaggat gggaaccatc caacgttcct gccttgtggc agcttcaagg
     6721 tggccacggt ggtattcgca ataatcttaa tactgagaat ccattatggg acgctattgt
     6781 tggcttagga ttcttgaagg acggtgtcaa aaatattcct tctttcttgt ctacggacaa
     6841 tatcggtact cgtgaaacat ttcttgctgg tctaattgat tctgatggct atgttactga
     6901 tgagcatggt attaaagcaa caataaagac aattcatact tctgtcagag atggtttggt
     6961 ttcccttgct cgttctttag gcttagtagt ctcggttaac gcagaacctg ctaaggttga
     7021 catgaatgtc accaaacata aaattagtta tgctatttat atgtctggtg gagatgtttt
     7081 gcttaacgtt ctttcgaagt gtgccggctc taaaaaattc aggcctgctc ccgccgctgc
     7141 ttttgcacgt gagtgccgcg gattttattt cgagttacaa gaattgaagg aagacgatta
     7201 ttatgggatt actttatctg atgattctga tcatcagttt ttgcttggat ctcaggttgt
     7261 tgtacagaac atgggctgtg acgataaatg cggctgtgcg gttccgtgtc cgggcggtac
     7321 gggctgtcgc tgtacctctg ctcgctcggg tgcggcggcg ggcgaacata ccacgtgcgg
     7381 ctgtggtgaa cactgcggtt gtaacccgtg cgcgtgtggc cgtgaaggca ccccgagcgg
     7441 ccgtgcgaat cgtcgcgcaa actgttcgtg cggtgcggcg tgtaactgtg cttcgtgcgg
     7501 ttcggcgacg gcataactgc aggtaattaa ataactagtt gatccggctg ctaacaaagc
     7561 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     7621 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggataact
     7681 acgtcaggtg gcacttttcg gggaa
//
