LOCUS       sequence-394907-        7583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            beta-domain of metallothionein 2a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7583)
  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 7583)
  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_beta-domain
FEATURES             Location/Qualifiers
     source          1..7583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..126
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(127..195,196..987)
                     /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(345..364)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1029..1542)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1224..1245)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1436..1455
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1653..2241
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2142..2161
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2395..2412
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2434..2453)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2594..2616
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2610..2801)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3358..4440)
                     /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     4402..4421
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4441..4518)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4671..4757
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4854..4940
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5037..5123
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5220..5306
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5403..5489
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5624..5643
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5624..5642
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5643..5667
                     /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             5682..5704
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5757..6575
                     /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             6582..6734
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6738..7286
                     /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(7484..7502)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7498..7545
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
       61 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
      121 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
      181 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
      241 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
      301 cgccccgaag aacgtttccc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      361 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
      421 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
      481 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
      541 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
      601 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
      661 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
      721 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
      781 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
      841 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
      901 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
      961 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     1021 attgatttac cccggttgat aatcagaaaa gccccaaaaa caggaagatt gtataagcaa
     1081 atatttaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     1141 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaatagc
     1201 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     1261 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     1321 cacccaaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     1381 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     1441 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     1501 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtaaaaggat ctaggtgaag
     1561 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     1621 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     1681 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     1741 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     1801 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     1861 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     1921 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     1981 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2041 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2101 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2161 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2221 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     2281 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     2341 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     2401 tcagtgagcg aggaagctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     2461 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     2521 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     2581 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     2641 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgca gcgattcaca gatgtctgcc
     2701 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     2761 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     2821 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     2881 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     2941 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagccgaa cgccagcaag
     3001 acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     3061 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     3121 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     3181 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     3241 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     3301 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     3361 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3421 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     3481 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     3541 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     3601 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
     3661 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     3721 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     3781 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     3841 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     3901 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     3961 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     4021 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     4081 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     4141 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     4201 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     4261 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     4321 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     4381 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     4441 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     4501 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     4561 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgcc
     4621 ggcatgccgc cctttcgtct tcaagaatta attcccaatt ccccaggcat caaataaaac
     4681 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     4741 tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag
     4801 ggtggcgggc aggacgcccg ccataaactg ccaggaatta attccccagg catcaaataa
     4861 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     4921 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     4981 gagggtggcg ggcaggacgc ccgccataaa ctgccaggaa ttaattcccc aggcatcaaa
     5041 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     5101 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc
     5161 ccggagggtg gcgggcagga cgcccgccat aaactgccag gaattaattc cccaggcatc
     5221 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     5281 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     5341 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggaattaa ttccccaggc
     5401 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     5461 cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc
     5521 aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggaat tggggatcgg
     5581 aattaattcc cggtttaaac cggggatctc gatcccgcga aattaatacg actcactata
     5641 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     5701 gagatatact tatgaaaatc gaagaaggta aactggtaat cggttccctg gagggttgct
     5761 ttgccaaggg taccaatgtt ttaatggcgg atgggtctat tgaatgtatt gaaaacattg
     5821 aggttggtaa taaggtcatg ggtaaagatg gcagacctcg tgaggtaatt aaattgccca
     5881 gaggaagaga aactatgtac agcgtcgtgc agaaaagtca gcacagagcc cacaaaagtg
     5941 actcaagtcg tgaagtgcca gaattactca agtttacgtg taatgcgacc catgagttgg
     6001 ttgttagaac acctcgtagt gtccgccgtt tgtctcgtac cattaagggt gtcgaatatt
     6061 ttgaagttat tacttttgag atgggccaaa agaaagcccc cgacggtaga attgttgagc
     6121 ttgtcaagga agtttcaaag agctacccaa tatctgaggg gcctgagaga gccaacgaat
     6181 tagtagaatc ctatagaaag gcttcaaata aagcttattt tgagtggact attgaggcca
     6241 gagatctttc tctgttgggt tcccatgttc gtaaagctac ctaccagact tacgctccaa
     6301 ttctttatga gaatgaccac tttttcgact acatgcaaaa aagtaagttt catctcacca
     6361 ttgaaggtcc aaaagtactt gcttatttac ttggtttatg gattggtgat ggattgtctg
     6421 acagggcaac tttttcggtt gattccagag atacttcttt gatggaacgt gttactgaat
     6481 atgctgaaaa gttgaatttg tgcgccgagt ataaggacag aaaagaacca caagttgcca
     6541 aaactgttaa tttgtactct aaagttgtca gaggtgctag cacaaatcct ggtgtatccg
     6601 cttggcaggt caacacagct tatactgcgg gacaattggt cacatataac ggcaagacgt
     6661 ataaatgttt gcagccccac acctccttgg caggatggga accatccaac gttcctgcct
     6721 tgtggcagct tcaaggtggc cacggtggta ttcgcaataa tcttaatact gagaatccat
     6781 tatgggacgc tattgttggc ttaggattct tgaaggacgg tgtcaaaaat attccttctt
     6841 tcttgtctac ggacaatatc ggtactcgtg aaacatttct tgctggtcta attgattctg
     6901 atggctatgt tactgatgag catggtatta aagcaacaat aaagacaatt catacttctg
     6961 tcagagatgg tttggtttcc cttgctcgtt ctttaggctt agtagtctcg gttaacgcag
     7021 aacctgctaa ggttgacatg aatgtcacca aacataaaat tagttatgct atttatatgt
     7081 ctggtggaga tgttttgctt aacgttcttt cgaagtgtgc cggctctaaa aaattcaggc
     7141 ctgctcccgc cgctgctttt gcacgtgagt gccgcggatt ttatttcgag ttacaagaat
     7201 tgaaggaaga cgattattat gggattactt tatctgatga ttctgatcat cagtttttgc
     7261 ttggatctca ggttgttgta cagaacatgg atccgaactg cagctgcgcg gcgggcgata
     7321 gctgcacctg cgcgggcagc tgcaaatgca aagaatgcaa atgcaccagc tgcaaaaaaa
     7381 gctaagatat cgtcgacgga tccgaattcc ctgcaggtaa ttaaataact agttgatccg
     7441 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     7501 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     7561 atatccggat aactacgtca ggt
//
