LOCUS       sequence-394827-        7673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant Oryza 
            sativa Metallothionein 1B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7673)
  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 7673)
  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: OsMTI-1B Oryza sativa Metallothionein 1B
FEATURES             Location/Qualifiers
     source          1..7673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        11..115
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(116..184,185..976)
                     /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(334..353)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1018..1531)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1213..1234)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1425..1444
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1642..2230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2131..2150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2384..2401
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2423..2442)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2583..2605
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2599..2790)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3347..4429)
                     /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     4391..4410
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4430..4507)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4660..4746
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4843..4929
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5026..5112
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5209..5295
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5392..5478
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5613..5632
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5613..5631
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5632..5656
                     /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             5671..5693
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5746..6564
                     /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             6571..6723
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6727..7275
                     /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(7563..7581)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7577..7624
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
       61 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
      121 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
      181 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
      241 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
      301 acgtttccca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
      361 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
      421 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
      481 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
      541 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
      601 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
      661 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
      721 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
      781 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
      841 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
      901 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
      961 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttacc
     1021 ccggttgata atcagaaaag ccccaaaaac aggaagattg tataagcaaa tatttaaatt
     1081 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     1141 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagcc cgagataggg
     1201 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     1261 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc acccaaatca
     1321 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     1381 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     1441 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     1501 gccgcgctta atgcgccgct acagggcgcg taaaaggatc taggtgaaga tcctttttga
     1561 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1621 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1681 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1741 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     1801 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1861 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1921 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1981 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2041 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2101 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2161 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2221 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     2281 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     2341 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     2401 ggaagctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     2461 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     2521 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     2581 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     2641 gcggtaaagc tcatcagcgt ggtcgtgcag cgattcacag atgtctgcct gttcatccgc
     2701 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     2761 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     2821 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     2881 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     2941 ggaccagaga aaaatcactc agggtcaatg ccagccgaac gccagcaaga cgtagcccag
     3001 cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     3061 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     3121 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     3181 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     3241 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     3301 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     3361 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3421 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     3481 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     3541 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     3601 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     3661 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     3721 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     3781 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     3841 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     3901 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     3961 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     4021 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     4081 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     4141 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     4201 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     4261 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     4321 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     4381 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     4441 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     4501 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     4561 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgccg gcatgccgcc
     4621 ctttcgtctt caagaattaa ttcccaattc cccaggcatc aaataaaacg aaaggctcag
     4681 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     4741 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     4801 ggacgcccgc cataaactgc caggaattaa ttccccaggc atcaaataaa acgaaaggct
     4861 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     4921 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggkggcgg
     4981 gcaggacgcc cgccataaac tgccaggaat taattcccca ggcatcaaat aaaacgaaag
     5041 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     5101 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     5161 cgggcaggac gcccgccata aactgccagg aattaattcc ccaggcatca aataaaacga
     5221 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     5281 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     5341 tggcgggcag gacgcccgcc ataaactgcc aggaattaat tccccaggca tcaaataaaa
     5401 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     5461 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     5521 gggtggcggg caggacgccc gccataaact gccaggaatt ggggatcgga attaattccc
     5581 ggtttaaacc ggggatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     5641 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatactt
     5701 atgaaaatcg aagaaggtaa actggtaatc ggttccctgg agggttgctt tgccaagggt
     5761 accaatgttt taatggcgga tgggtctatt gaatgtattg aaaacattga ggttggtaat
     5821 aaggtcatgg gtaaagatgg cagacctcgt gaggtaatta aattgcccag aggaagagaa
     5881 actatgtaca gcgtcgtgca gaaaagtcag cacagagccc acaaaagtga ctcaagtcgt
     5941 gaagtgccag aattactcaa gtttacgtgt aatgcgaccc atgagttggt tgttagaaca
     6001 cctcgtagtg tccgccgttt gtctcgtacc attaagggtg tcgaatattt tgaagttatt
     6061 acttttgaga tgggccaaaa gaaagccccc gacggtagaa ttgttgagct tgtcaaggaa
     6121 gtttcaaaga gctacccaat atctgagggg cctgagagag ccaacgaatt agtagaatcc
     6181 tatagaaagg cttcaaataa agcttatttt gagtggacta ttgaggccag agatctttct
     6241 ctgttgggtt cccatgttcg taaagctacc taccagactt acgctccaat tctttatgag
     6301 aatgaccact ttttcgacta catgcaaaaa agtaagtttc atctcaccat tgaaggtcca
     6361 aaagtacttg cttatttact tggtttatgg attggtgatg gattgtctga cagggcaact
     6421 ttttcggttg attccagaga tacttctttg atggaacgtg ttactgaata tgctgaaaag
     6481 ttgaatttgt gcgccgagta taaggacaga aaagaaccac aagttgccaa aactgttaat
     6541 ttgtactcta aagttgtcag aggtgctagc acaaatcctg gtgtatccgc ttggcaggtc
     6601 aacacagctt atactgcggg acaattggtc acatataacg gcaagacgta taaatgtttg
     6661 cagccccaca cctccttggc aggatgggaa ccatccaacg ttcctgcctt gtggcagctt
     6721 caaggtggcc acggtggtat tcgcaataat cttaatactg agaatccatt atgggacgct
     6781 attgttggct taggattctt gaaggacggt gtcaaaaata ttccttcttt cttgtctacg
     6841 gacaatatcg gtactcgtga aacatttctt gctggtctaa ttgattctga tggctatgtt
     6901 actgatgagc atggtattaa agcaacaata aagacaattc atacttctgt cagagatggt
     6961 ttggtttccc ttgctcgttc tttaggctta gtagtctcgg ttaacgcaga acctgctaag
     7021 gttgacatga atgtcaccaa acataaaatt agttatgcta tttatatgtc tggtggagat
     7081 gttttgctta acgttctttc gaagtgtgcc ggctctaaaa aattcaggcc tgctcccgcc
     7141 gctgcttttg cacgtgagtg ccgcggattt tatttcgagt tacaagaatt gaaggaagac
     7201 gattattatg ggattacttt atctgatgat tctgatcatc agtttttgct tggatctcag
     7261 gttgttgtac agaacatgtc ttgctcttgc ggttctagct gcggttgtgg ttccaactgt
     7321 acctgcggca aaatgtatcc tgacctggaa gaaaaatcct cttccgctca agcgactgtt
     7381 gtactgggcg tagccccgga aaaagcacac tttgaggctg cagcagagtc tggtgaaacg
     7441 gctcacggtt gcggctgcgg ctcctcttgc aagtgcaacc cgtgcaactg ctaaggtaat
     7501 taaataacta gttgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     7561 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     7621 tttgctgaaa ggaggaacta tatccggata actacgtcag gtggcacttt tcg
//
