LOCUS       sequence-203314-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 2a C21A mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7130)
  AUTHORS   Singh AK, Pomorski A, Wu S, Peris-Diaz MD, Czepczynska-Krezel H, 
            Krezel A
  TITLE     The connection of alpha- and beta-domains in mammalian 
            metallothionein-2 differentiates Zn(II) binding affinities, affects 
            folding, and determines zinc buffering properties.
  JOURNAL   Metallomics. 2023 Jun 1;15(6):mfad029. doi: 10.1093/mtomcs/mfad029.
  PUBMED    37147085
REFERENCE   2  (bases 1 to 7130)
  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: "2023-06-1"; volume: "15(6):mfad029. doi"; 
            pages: " 10.1093/mtomcs/mfad029"
COMMENT     Sequence Label: MT2a_C21A
FEATURES             Location/Qualifiers
     source          1..7130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        4..108
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(109..177,178..969)
                     /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(327..346)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1011..1524)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1206..1227)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1418..1437
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1635..2223
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2124..2143
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2377..2394
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2416..2435)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2576..2598
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2592..2783)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3340..4422)
                     /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     4384..4403
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4423..4500)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4653..4739
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4836..4922
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5057..5076
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5057..5075
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5076..5100
                     /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             5115..5137
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5190..6008
                     /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             6015..6167
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6171..6719
                     /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(7013..7031)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7027..7074
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
       61 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
      121 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
      181 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
      241 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttc
      301 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc
      361 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
      421 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
      481 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
      541 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
      601 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
      661 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
      721 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
      781 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
      841 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
      901 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
      961 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt accccggttg
     1021 ataatcagaa aagccccaaa aacaggaaga ttgtataagc aaatatttaa attgtaaacg
     1081 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     1141 aggccgaaat cggcaaaatc ccttataaat caaaagaata gcccgagata gggttgagtg
     1201 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     1261 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcacccaaa tcaagttttt
     1321 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     1381 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     1441 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     1501 ttaatgcgcc gctacagggc gcgtaaaagg atctaggtga agatcctttt tgataatctc
     1561 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1621 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1681 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1741 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     1801 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1861 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     1921 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1981 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2041 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2101 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     2161 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     2221 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     2281 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     2341 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagct
     2401 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     2461 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     2521 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     2581 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     2641 agctcatcag cgtggtcgtg cagcgattca cagatgtctg cctgttcatc cgcgtccagc
     2701 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     2761 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     2821 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     2881 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     2941 agaaaaatca ctcagggtca atgccagccg aacgccagca agacgtagcc cagcgcgtcg
     3001 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     3061 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     3121 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     3181 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     3241 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     3301 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3361 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3421 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     3481 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     3541 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     3601 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     3661 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     3721 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     3781 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     3841 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     3901 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     3961 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     4021 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     4081 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     4141 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     4201 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     4261 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     4321 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     4381 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     4441 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     4501 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     4561 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg ccggcatgcc gccctttcgt
     4621 cttcaagaat taattcccaa ttccccaggc atcaaataaa acgaaaggct cagtcgaaag
     4681 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
     4741 cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc
     4801 cgccataaac tgccaggaat taattcccca ggcatcaaat aaaacgaaag gctcagtcga
     4861 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
     4921 atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac
     4981 gcccgccata aactgccagg aattggggat cggaattaat tcccggttta aaccggggat
     5041 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     5101 cctctagaaa taattttgtt taactttaag aaggagatat acttatgaaa atcgaagaag
     5161 gtaaactggt aatcggttcc ctggagggtt gctttgccaa gggtaccaat gttttaatgg
     5221 cggatggctc tattgaatgt attgaaaaca ttgaggttgg taataaggtc atgggtaaag
     5281 atggcagacc tcgtgaggta attaaattgc ccagaggaag agaaactatg tacagcgtcg
     5341 tgcagaaaag tcagcacaga gcccacaaaa gtgactcaag tcgtgaagtg ccagaattac
     5401 tcaagtttac gtgtaatgcg acccatgagt tggttgttag aacacctcgt agtgtccgcc
     5461 gtttgtctcg taccattaag ggtgtcgaat attttgaagt tattactttt gagatgggcc
     5521 aaaagaaagc ccccgacggt agaattgttg agcttgtcaa ggaagtttca aagagctacc
     5581 caatatctga ggggcctgag agagccaacg aattagtaga atcctataga aaggcttcaa
     5641 ataaagctta ttttgagtgg actattgagg ccagagatct ttctctgttg ggttcccatg
     5701 ttcgtaaagc tacctaccag acttacgctc caattcttta tgagaatgac cactttttcg
     5761 actacatgca aaaaagtaag tttcatctca ccattgaagg tccaaaagta cttgcttatt
     5821 tacttggttt atggattggt gatggattgt ctgacagggc aactttttcg gttgattcca
     5881 gagatacttc tttgatggaa cgtgttactg aatatgctga aaagttgaat ttgtgcgccg
     5941 agtataagga cagaaaagaa ccacaagttg ccaaaactgt taatttgtac tctaaagttg
     6001 tcagaggtgc tagcacaaat cctggtgtat ccgcttggca ggtcaacaca gcttatactg
     6061 cgggacaatt ggtcacatat aacggcaaga cgtataaatg tttgcagccc cacacctcct
     6121 tggcaggatg ggaaccatcc aacgttcctg ccttgtggca gcttcaaggt ggccacggtg
     6181 gtattcgcaa taatcttaat actgagaatc cattatggga cgctattgtt ggcttaggat
     6241 tcttgaagga cggtgtcaaa aatattcctt ctttcttgtc tacggacaat atcggtactc
     6301 gtgaaacatt tcttgctggt ctaattgatt ctgatggcta tgttactgat gagcatggta
     6361 ttaaagcaac aataaagaca attcatactt ctgtcagaga tggtttggtt tcccttgctc
     6421 gttctttagg cttagtagtc tcggttaacg cagaacctgc taaggttgac atgaatgtca
     6481 ccaaacataa aattagttat gctatttata tgtctggtgg agatgttttg cttaacgttc
     6541 tttcgaagtg tgccggctct aaaaaattca ggcctgctcc cgccgctgct tttgcacgtg
     6601 agtgccgcgg attttatttc gagttacaag aattgaagga agacgattat tatgggatta
     6661 ctttatctga tgattctgat catcagtttt tgcttggatc tcaggttgtt gtacagaaca
     6721 tggatccgaa ctgcagctgt gcggcgggtg attcttgcac gtgtgcgggc agctgcaaag
     6781 cgaaagaatg caaatgcacc agctgcaaaa aaagctgctg cagctgctgt ccggtgggct
     6841 gtgcgaaatg cgcgcaaggc tgcatttgca aaggcgcgag cgataaatgc agctgctgcg
     6901 cgtaactgca gaccaccatc gtcgacggat ccgaattccc tgcaggtaat taaataacta
     6961 gttgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     7021 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     7081 ggaggaacta tatccggata actacgtcag gtggcacttt tcggggaaat
//
