LOCUS       sequence-203719-        7117 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7117)
  AUTHORS   Peris-Diaz MD, Guran R, Zitka O, Adam V, Krezel A
  TITLE     Mass Spectrometry-Based Structural Analysis of Cysteine-Rich 
            Metal-Binding Sites in Proteins with MetaOdysseus R Software.
  JOURNAL   J Proteome Res. 2021 Jan 1;20(1):776-785. doi: 
            10.1021/acs.jproteome.0c00651. Epub 2020 Sep 28.
  PUBMED    32924499
REFERENCE   2  (bases 1 to 7117)
  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: 
            "10.1021/acs.jproteome.0c00651"; journalName: "J Proteome Res"; 
            date: "2021-01-1- 1"; volume: "20"; issue: "1"; pages: "776-785"
COMMENT     Sequence Label: MT3
FEATURES             Location/Qualifiers
     source          1..7117
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     500..519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     753..770
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(792..811)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     952..974
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(968..1159)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1716..2798)
                     /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     2760..2779
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2799..2876)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      3029..3115
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3212..3298
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3433..3452
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3433..3451
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3452..3476
                     /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             3491..3513
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3566..4384
                     /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             4391..4543
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             4547..5095
                     /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(5376..5394)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5390..5437
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        5497..5601
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5602..5670,5671..6462)
                     /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(5820..5839)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6504..7017)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6699..6720)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6911..6930
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
       61 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
      121 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
      181 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
      241 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
      301 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
      361 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
      421 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
      481 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
      541 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      601 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
      661 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
      721 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagctatgg
      781 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
      841 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
      901 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
      961 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     1021 catcagcgtg gtcgtgcagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     1081 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     1141 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     1201 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     1261 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     1321 aaatcactca gggtcaatgc cagccgaacg ccagcaagac gtagcccagc gcgtcggccg
     1381 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1441 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1501 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1561 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1621 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1681 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     1741 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     1801 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     1861 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     1921 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     1981 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2041 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2101 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2161 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2221 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2281 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2341 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2401 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2461 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2521 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2581 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2641 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     2701 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     2761 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     2821 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     2881 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     2941 gccgttgagc accgccgccg caaggaatgg tgcatgccgg catgccgccc tttcgtcttc
     3001 aagaattaat tcccaattcc ccaggcatca aataaaacga aaggctcagt cgaaagactg
     3061 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     3121 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     3181 ataaactgcc aggaattaat tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     3241 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     3301 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc
     3361 gccataaact gccaggaatt ggggatcgga attaattccc ggtttaaacc ggggatctcg
     3421 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     3481 tagaaataat tttgtttaac tttaagaagg agatatactt atgaaaatcg aagaaggtaa
     3541 actggtaatc ggttccctgg agggttgctt tgccaagggt accaatgttt taatggcgga
     3601 tgggtctatt gaatgtattg aaaacattga ggttggtaat aaggtcatgg gtaaagatgg
     3661 cagacctcgt gaggtaatta aattgcccag aggaagagaa actatgtaca gcgtcgtgca
     3721 gaaaagtcag cacagagccc acaaaagtga ctcaagtcgt gaagtgccag aattactcaa
     3781 gtttacgtgt aatgcgaccc atgagttggt tgttagaaca cctcgtagtg tccgccgttt
     3841 gtctcgtacc attaagggtg tcgaatattt tgaagttatt acttttgaga tgggccaaaa
     3901 gaaagccccc gacggtagaa ttgttgagct tgtcaaggaa gtttcaaaga gctacccaat
     3961 atctgagggg cctgagagag ccaacgaatt agtagaatcc tatagaaagg cttcaaataa
     4021 agcttatttt gagtggacta ttgaggccag agatctttct ctgttgggtt cccatgttcg
     4081 taaagctacc taccagactt acgctccaat tctttatgag aatgaccact ttttcgacta
     4141 catgcaaaaa agtaagtttc atctcaccat tgaaggtcca aaagtacttg cttatttact
     4201 tggtttatgg attggtgatg gattgtctga cagggcaact ttttcggttg attccagaga
     4261 tacttctttg atggaacgtg ttactgaata tgctgaaaag ttgaatttgt gcgccgagta
     4321 taaggacaga aaagaaccac aagttgccaa aactgttaat ttgtactcta aagttgtcag
     4381 aggtgctagc acaaatcctg gtgtatccgc ttggcaggtc aacacagctt atactgcggg
     4441 acaattggtc acatataacg gcaagacgta taaatgtttg cagccccaca cctccttggc
     4501 aggatgggaa ccatccaacg ttcctgcctt gtggcagctt caaggtggcc acggtggtat
     4561 tcgcaataat cttaatactg agaatccatt atgggacgct attgttggct taggattctt
     4621 gaaggacggt gtcaaaaata ttccttcttt cttgtctacg gacaatatcg gtactcgtga
     4681 aacatttctt gctggtctaa ttgattctga tggctatgtt actgatgagc atggtattaa
     4741 agcaacaata aagacaattc atacttctgt cagagatggt ttggtttccc ttgctcgttc
     4801 tttaggctta gtagtctcgg ttaacgcaga acctgctaag gttgacatga atgtcaccaa
     4861 acataaaatt agttatgcta tttatatgtc tggtggagat gttttgctta acgttctttc
     4921 gaagtgtgcc ggctctaaaa aattcaggcc tgctcccgcc gctgcttttg cacgtgagtg
     4981 ccgcggattt tatttcgagt tacaagaatt gaaggaagac gattattatg ggattacttt
     5041 atctgatgat tctgatcatc agtttttgct tggatctcag gttgttgtac agaacatgga
     5101 cccggaaacc tgcccgtgcc cgtctggtgg ctcctgtacc tgtgccgatt cgtgtaaatg
     5161 tgaaggctgt aaatgtacct catgcaaaaa gagctgctgt tcttgctgtc cggcggaatg
     5221 cgaaaaatgt gcaaaggatt gcgtgtgtaa aggcggtgaa gcggccgaag cagaagctga
     5281 aaagtgtagt tgctgtcagt aactgcaggt aattaaataa ctagttgatc cggctgctaa
     5341 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     5401 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     5461 ataactacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5521 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5581 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5641 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5701 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5761 atccttgaga gttttcgccc cgaagaacgt ttcccaatga tgagcacttt taaagttctg
     5821 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     5881 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5941 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6001 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6061 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6121 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6181 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6241 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6301 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6361 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6421 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6481 tcatatatac tttagattga tttaccccgg ttgataatca gaaaagcccc aaaaacagga
     6541 agattgtata agcaaatatt taaattgtaa acgttaatat tttgttaaaa ttcgcgttaa
     6601 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     6661 aatcaaaaga atagcccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     6721 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     6781 cactacgtga accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     6841 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     6901 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     6961 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtaaa
     7021 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     7081 tcgttccact gagcgtcaga ccccgtagaa aagatca
//
