LOCUS       sequence-394924-        7628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            Thermosynechococcus vulcanus Metallothionein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7628)
  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 7628)
  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: TvMT Thermosynechococcus vulcanus Metallothionein
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        17..121
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(122..190,191..982)
                     /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(340..359)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1024..1537)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1219..1240)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1431..1450
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1648..2236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2137..2156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2390..2407
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2429..2448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2589..2611
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2605..2796)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3353..4435)
                     /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     4397..4416
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4436..4513)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4666..4752
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4849..4935
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5032..5118
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5215..5301
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5398..5484
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5619..5638
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5619..5637
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5638..5662
                     /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             5677..5699
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5752..6570
                     /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             6577..6729
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6733..7281
                     /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(7524..7542)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7538..7585
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
       61 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
      121 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
      181 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
      241 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
      301 cgaagaacgt ttcccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
      361 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
      421 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
      481 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
      541 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
      601 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
      661 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
      721 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
      781 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
      841 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
      901 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
      961 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     1021 tttaccccgg ttgataatca gaaaagcccc aaaaacagga agattgtata agcaaatatt
     1081 taaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     1141 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagcccgag
     1201 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     1261 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     1321 aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     1381 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     1441 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     1501 acacccgccg cgcttaatgc gccgctacag ggcgcgtaaa aggatctagg tgaagatcct
     1561 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1621 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1681 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1741 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     1801 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1861 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1921 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1981 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2041 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2101 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2161 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2221 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     2281 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2341 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     2401 gagcgaggaa gctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     2461 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     2521 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     2581 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     2641 gcagctgcgg taaagctcat cagcgtggtc gtgcagcgat tcacagatgt ctgcctgttc
     2701 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     2761 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     2821 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     2881 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     2941 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag ccgaacgcca gcaagacgta
     3001 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     3061 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     3121 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     3181 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     3241 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     3301 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     3361 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3421 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     3481 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     3541 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     3601 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
     3661 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     3721 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     3781 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     3841 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     3901 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     3961 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     4021 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     4081 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     4141 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     4201 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     4261 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     4321 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     4381 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     4441 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     4501 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     4561 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgccggcat
     4621 gccgcccttt cgtcttcaag aattaattcc caattcccca ggcatcaaat aaaacgaaag
     4681 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     4741 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     4801 cgggcaggac gcccgccata aactgccagg aattaattcc ccaggcatca aataaaacga
     4861 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     4921 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     4981 tggcgggcag gacgcccgcc ataaactgcc aggaattaat tccccaggca tcaaataaaa
     5041 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     5101 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     5161 gggtggcggg caggacgccc gccataaact gccaggaatt aattccccag gcatcaaata
     5221 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     5281 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
     5341 ggagggtggc gggcaggacg cccgccataa actgccagga attaattccc caggcatcaa
     5401 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     5461 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
     5521 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggaattgggg atcggaatta
     5581 attcccggtt taaaccgggg atctcgatcc cgcgaaatta atacgactca ctatagggga
     5641 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     5701 atacttatga aaatcgaaga aggtaaactg gtaatcggtt ccctggaggg ttgctttgcc
     5761 aagggtacca atgttttaat ggcggatggg tctattgaat gtattgaaaa cattgaggtt
     5821 ggtaataagg tcatgggtaa agatggcaga cctcgtgagg taattaaatt gcccagagga
     5881 agagaaacta tgtacagcgt cgtgcagaaa agtcagcaca gagcccacaa aagtgactca
     5941 agtcgtgaag tgccagaatt actcaagttt acgtgtaatg cgacccatga gttggttgtt
     6001 agaacacctc gtagtgtccg ccgtttgtct cgtaccatta agggtgtcga atattttgaa
     6061 gttattactt ttgagatggg ccaaaagaaa gcccccgacg gtagaattgt tgagcttgtc
     6121 aaggaagttt caaagagcta cccaatatct gaggggcctg agagagccaa cgaattagta
     6181 gaatcctata gaaaggcttc aaataaagct tattttgagt ggactattga ggccagagat
     6241 ctttctctgt tgggttccca tgttcgtaaa gctacctacc agacttacgc tccaattctt
     6301 tatgagaatg accacttttt cgactacatg caaaaaagta agtttcatct caccattgaa
     6361 ggtccaaaag tacttgctta tttacttggt ttatggattg gtgatggatt gtctgacagg
     6421 gcaacttttt cggttgattc cagagatact tctttgatgg aacgtgttac tgaatatgct
     6481 gaaaagttga atttgtgcgc cgagtataag gacagaaaag aaccacaagt tgccaaaact
     6541 gttaatttgt actctaaagt tgtcagaggt gctagcacaa atcctggtgt atccgcttgg
     6601 caggtcaaca cagcttatac tgcgggacaa ttggtcacat ataacggcaa gacgtataaa
     6661 tgtttgcagc cccacacctc cttggcagga tgggaaccat ccaacgttcc tgccttgtgg
     6721 cagcttcaag gtggccacgg tggtattcgc aataatctta atactgagaa tccattatgg
     6781 gacgctattg ttggcttagg attcttgaag gacggtgtca aaaatattcc ttctttcttg
     6841 tctacggaca atatcggtac tcgtgaaaca tttcttgctg gtctaattga ttctgatggc
     6901 tatgttactg atgagcatgg tattaaagca acaataaaga caattcatac ttctgtcaga
     6961 gatggtttgg tttcccttgc tcgttcttta ggcttagtag tctcggttaa cgcagaacct
     7021 gctaaggttg acatgaatgt caccaaacat aaaattagtt atgctattta tatgtctggt
     7081 ggagatgttt tgcttaacgt tctttcgaag tgtgccggct ctaaaaaatt caggcctgct
     7141 cccgccgctg cttttgcacg tgagtgccgc ggattttatt tcgagttaca agaattgaag
     7201 gaagacgatt attatgggat tactttatct gatgattctg atcatcagtt tttgcttgga
     7261 tctcaggttg ttgtacagaa catgactacc gttacccaga tgaaatgtgc ttgcccgcat
     7321 tgcctgtgca tcgtgtctct gaacgatgca attatggtcg atggcaaacc gtattgttcc
     7381 gaagtgtgtg caaacggcac ttgcaaagaa aactccggct gtggccatgc tggttgcggt
     7441 tgcggttccg cataaggtaa ttaaataact agttgatccg gctgctaaca aagcccgaaa
     7501 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     7561 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat aactacgtca
     7621 ggtggcac
//
