LOCUS       sequence-394816-        7654 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression plasmids for production of recombinant Purple 
            Sea Urchin Strongylocentrotus purpuratus Metallothionein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7654)
  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 7654)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: SpMTA Purple Sea Urchin Metallothionein
FEATURES             Location/Qualifiers
     source          1..7654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        43..147
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(148..216,217..1008)
                     /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(366..385)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1050..1563)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1245..1266)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1457..1476
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1674..2262
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2163..2182
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2416..2433
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2455..2474)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2615..2637
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2631..2822)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3379..4461)
                     /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     4423..4442
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4462..4539)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4692..4778
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4875..4961
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5058..5144
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5241..5327
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5424..5510
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5645..5664
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5645..5663
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5664..5688
                     /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             5703..5725
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5778..6596
                     /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             6603..6755
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6759..7307
                     /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(7576..7594)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7590..7637
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 atccggataa ctacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
       61 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
      121 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
      181 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
      241 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
      301 ggtaagatcc ttgagagttt tcgccccgaa gaacgtttcc caatgatgag cacttttaaa
      361 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
      421 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
      481 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
      541 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
      601 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
      661 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
      721 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
      781 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
      841 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
      901 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
      961 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     1021 gtttactcat atatacttta gattgattta ccccggttga taatcagaaa agccccaaaa
     1081 acaggaagat tgtataagca aatatttaaa ttgtaaacgt taatattttg ttaaaattcg
     1141 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     1201 cttataaatc aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga
     1261 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     1321 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag
     1381 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     1441 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     1501 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     1561 cgtaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     1621 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1681 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1741 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1801 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     1861 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1921 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     1981 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2041 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     2101 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     2161 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2221 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2281 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     2341 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     2401 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcta tggtgcactc tcagtacaat
     2461 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     2521 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2581 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2641 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc
     2701 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     2761 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     2821 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     2881 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     2941 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3001 tgccagccga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     3061 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     3121 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     3181 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     3241 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     3301 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     3361 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3421 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     3481 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     3541 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     3601 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     3661 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     3721 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     3781 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     3841 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     3901 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     3961 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     4021 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     4081 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     4141 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     4201 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     4261 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     4321 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     4381 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     4441 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     4501 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     4561 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     4621 ccgcaaggaa tggtgcatgc cggcatgccg ccctttcgtc ttcaagaatt aattcccaat
     4681 tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     4741 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     4801 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggaatt
     4861 aattccccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     4921 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     4981 acgttgcgaa gcaacggccc ggagggkggc gggcaggacg cccgccataa actgccagga
     5041 attaattccc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     5101 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     5161 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     5221 ggaattaatt ccccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     5281 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     5341 atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     5401 ccaggaatta attccccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     5461 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag
     5521 cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa
     5581 ctgccaggaa ttggggatcg gaattaattc ccggtttaaa ccggggatct cgatcccgcg
     5641 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     5701 attttgttta actttaagaa ggagatatac ttatgaaaat cgaagaaggt aaactggtaa
     5761 tcggttccct ggagggttgc tttgccaagg gtaccaatgt tttaatggcg gatgggtcta
     5821 ttgaatgtat tgaaaacatt gaggttggta ataaggtcat gggtaaagat ggcagacctc
     5881 gtgaggtaat taaattgccc agaggaagag aaactatgta cagcgtcgtg cagaaaagtc
     5941 agcacagagc ccacaaaagt gactcaagtc gtgaagtgcc agaattactc aagtttacgt
     6001 gtaatgcgac ccatgagttg gttgttagaa cacctcgtag tgtccgccgt ttgtctcgta
     6061 ccattaaggg tgtcgaatat tttgaagtta ttacttttga gatgggccaa aagaaagccc
     6121 ccgacggtag aattgttgag cttgtcaagg aagtttcaaa gagctaccca atatctgagg
     6181 ggcctgagag agccaacgaa ttagtagaat cctatagaaa ggcttcaaat aaagcttatt
     6241 ttgagtggac tattgaggcc agagatcttt ctctgttggg ttcccatgtt cgtaaagcta
     6301 cctaccagac ttacgctcca attctttatg agaatgacca ctttttcgac tacatgcaaa
     6361 aaagtaagtt tcatctcacc attgaaggtc caaaagtact tgcttattta cttggtttat
     6421 ggattggtga tggattgtct gacagggcaa ctttttcggt tgattccaga gatacttctt
     6481 tgatggaacg tgttactgaa tatgctgaaa agttgaattt gtgcgccgag tataaggaca
     6541 gaaaagaacc acaagttgcc aaaactgtta atttgtactc taaagttgtc agaggtgcta
     6601 gcacaaatcc tggtgtatcc gcttggcagg tcaacacagc ttatactgcg ggacaattgg
     6661 tcacatataa cggcaagacg tataaatgtt tgcagcccca cacctccttg gcaggatggg
     6721 aaccatccaa cgttcctgcc ttgtggcagc ttcaaggtgg ccacggtggt attcgcaata
     6781 atcttaatac tgagaatcca ttatgggacg ctattgttgg cttaggattc ttgaaggacg
     6841 gtgtcaaaaa tattccttct ttcttgtcta cggacaatat cggtactcgt gaaacatttc
     6901 ttgctggtct aattgattct gatggctatg ttactgatga gcatggtatt aaagcaacaa
     6961 taaagacaat tcatacttct gtcagagatg gtttggtttc ccttgctcgt tctttaggct
     7021 tagtagtctc ggttaacgca gaacctgcta aggttgacat gaatgtcacc aaacataaaa
     7081 ttagttatgc tatttatatg tctggtggag atgttttgct taacgttctt tcgaagtgtg
     7141 ccggctctaa aaaattcagg cctgctcccg ccgctgcttt tgcacgtgag tgccgcggat
     7201 tttatttcga gttacaagaa ttgaaggaag acgattatta tgggattact ttatctgatg
     7261 attctgatca tcagtttttg cttggatctc aggttgttgt acagaacatg ccggatgtga
     7321 aatgtgtgtg ctgtaccgaa ggcaaagaat gtgcttgttt tggtcaggat tgttgtgtta
     7381 cgggcgaatg ctgcaaagat ggcacctgct gtggtatttg cacgaacgcg gcctgcaaat
     7441 gtgcaaatgg ctgcaaatgt ggcagcggtt gctcttgtac cgagggtaac tgcgcgtgtt
     7501 aactgcaggt aattaaataa ctagttgatc cggctgctaa caaagcccga aaggaagctg
     7561 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     7621 tcttgagggg ttttttgctg aaaggaggaa ctat
//
