LOCUS       sequence-394923-        7643 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant Xenopus 
            laevis Metallothionein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7643)
  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 7643)
  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: XlMT Xenopus laevis Metallothionein
FEATURES             Location/Qualifiers
     source          1..7643
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        31..135
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(136..204,205..996)
                     /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(354..373)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1038..1551)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1233..1254)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1445..1464
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1662..2250
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2151..2170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2404..2421
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2443..2462)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2603..2625
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2619..2810)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3367..4449)
                     /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     4411..4430
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4450..4527)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4680..4766
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4863..4949
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5046..5132
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5229..5315
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5412..5498
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5633..5652
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5633..5651
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5652..5676
                     /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             5691..5713
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5766..6584
                     /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             6591..6743
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6747..7295
                     /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(7553..7571)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7567..7614
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
       61 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
      121 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
      181 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
      241 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
      301 gagagttttc gccccgaaga acgtttccca atgatgagca cttttaaagt tctgctatgt
      361 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
      421 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
      481 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
      541 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
      601 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
      661 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
      721 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
      781 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
      841 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
      901 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
      961 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     1021 atactttaga ttgatttacc ccggttgata atcagaaaag ccccaaaaac aggaagattg
     1081 tataagcaaa tatttaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt
     1141 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     1201 aagaatagcc cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     1261 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     1321 gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     1381 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     1441 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     1501 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg taaaaggatc
     1561 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     1621 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1681 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1741 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1801 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1861 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1921 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1981 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2041 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2101 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2161 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2221 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2281 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2341 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     2401 cgcagcgagt cagtgagcga ggaagctatg gtgcactctc agtacaatct gctctgatgc
     2461 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     2521 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     2581 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     2641 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgcag cgattcacag
     2701 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     2761 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     2821 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     2881 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     2941 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagccgaac
     3001 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
     3061 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     3121 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     3181 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     3241 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     3301 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     3361 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3421 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     3481 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     3541 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     3601 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac
     3661 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     3721 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     3781 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     3841 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     3901 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     3961 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     4021 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     4081 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     4141 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     4201 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     4261 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     4321 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     4381 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     4441 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     4501 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     4561 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     4621 gtgcatgccg gcatgccgcc ctttcgtctt caagaattaa ttcccaattc cccaggcatc
     4681 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     4741 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     4801 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggaattaa ttccccaggc
     4861 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     4921 cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc
     4981 aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggaat taattcccca
     5041 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     5101 tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga
     5161 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg aattaattcc
     5221 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     5281 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     5341 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggaattaat
     5401 tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     5461 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     5521 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggaatt
     5581 ggggatcgga attaattccc ggtttaaacc ggggatctcg atcccgcgaa attaatacga
     5641 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac
     5701 tttaagaagg agatatactt atgaaaatcg aagaaggtaa actggtaatc ggttccctgg
     5761 agggttgctt tgccaagggt accaatgttt taatggcgga tgggtctatt gaatgtattg
     5821 aaaacattga ggttggtaat aaggtcatgg gtaaagatgg cagacctcgt gaggtaatta
     5881 aattgcccag aggaagagaa actatgtaca gcgtcgtgca gaaaagtcag cacagagccc
     5941 acaaaagtga ctcaagtcgt gaagtgccag aattactcaa gtttacgtgt aatgcgaccc
     6001 atgagttggt tgttagaaca cctcgtagtg tccgccgttt gtctcgtacc attaagggtg
     6061 tcgaatattt tgaagttatt acttttgaga tgggccaaaa gaaagccccc gacggtagaa
     6121 ttgttgagct tgtcaaggaa gtttcaaaga gctacccaat atctgagggg cctgagagag
     6181 ccaacgaatt agtagaatcc tatagaaagg cttcaaataa agcttatttt gagtggacta
     6241 ttgaggccag agatctttct ctgttgggtt cccatgttcg taaagctacc taccagactt
     6301 acgctccaat tctttatgag aatgaccact ttttcgacta catgcaaaaa agtaagtttc
     6361 atctcaccat tgaaggtcca aaagtacttg cttatttact tggtttatgg attggtgatg
     6421 gattgtctga cagggcaact ttttcggttg attccagaga tacttctttg atggaacgtg
     6481 ttactgaata tgctgaaaag ttgaatttgt gcgccgagta taaggacaga aaagaaccac
     6541 aagttgccaa aactgttaat ttgtactcta aagttgtcag aggtgctagc acaaatcctg
     6601 gtgtatccgc ttggcaggtc aacacagctt atactgcggg acaattggtc acatataacg
     6661 gcaagacgta taaatgtttg cagccccaca cctccttggc aggatgggaa ccatccaacg
     6721 ttcctgcctt gtggcagctt caaggtggcc acggtggtat tcgcaataat cttaatactg
     6781 agaatccatt atgggacgct attgttggct taggattctt gaaggacggt gtcaaaaata
     6841 ttccttcttt cttgtctacg gacaatatcg gtactcgtga aacatttctt gctggtctaa
     6901 ttgattctga tggctatgtt actgatgagc atggtattaa agcaacaata aagacaattc
     6961 atacttctgt cagagatggt ttggtttccc ttgctcgttc tttaggctta gtagtctcgg
     7021 ttaacgcaga acctgctaag gttgacatga atgtcaccaa acataaaatt agttatgcta
     7081 tttatatgtc tggtggagat gttttgctta acgttctttc gaagtgtgcc ggctctaaaa
     7141 aattcaggcc tgctcccgcc gctgcttttg cacgtgagtg ccgcggattt tatttcgagt
     7201 tacaagaatt gaaggaagac gattattatg ggattacttt atctgatgat tctgatcatc
     7261 agtttttgct tggatctcag gttgttgtac agaacatgga cccgcaagac tgtaaatgtg
     7321 aaacgggcgc atcctgttcc tgtggcacga cctgtagctg ctcaaactgt aaatgtacct
     7381 catgcaaaaa atcttgctgt tcttgctgtc cggcggaatg cagtaaatgt tcccagggct
     7441 gccattgtga aaaaggtagc aaaaaatgta gctgctgtaa ctaaggtaat taaataacta
     7501 gttgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     7561 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     7621 ggaggaacta tatccggata act
//
