LOCUS       sequence-203330-        7099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 3 subisoform with C30Y and R31W substitution.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7099)
  TITLE     Krezel Lab Plasmids
REFERENCE   2  (bases 1 to 7099)
  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"
COMMENT     Sequence Label: MT4_C30Y R31W
FEATURES             Location/Qualifiers
     source          1..7099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..118
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(119..187,188..979)
                     /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(337..356)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1021..1534)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1216..1237)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1428..1447
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1645..2233
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2134..2153
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2387..2404
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2426..2445)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2586..2608
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2602..2793)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3350..4432)
                     /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     4394..4413
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4433..4510)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4663..4749
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4846..4932
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5067..5086
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5067..5085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5086..5110
                     /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             5125..5147
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5200..6018
                     /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             6025..6177
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6181..6729
                     /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(6992..7010)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7006..7053
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
       61 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      121 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      181 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
      241 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
      301 agaacgtttc ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      361 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      421 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      481 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      541 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      601 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      661 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      721 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      781 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      841 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      901 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      961 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1021 accccggttg ataatcagaa aagccccaaa aacaggaaga ttgtataagc aaatatttaa
     1081 attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     1141 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gcccgagata
     1201 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     1261 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcacccaaa
     1321 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     1381 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     1441 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     1501 cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg atctaggtga agatcctttt
     1561 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1621 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1681 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1741 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     1801 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1861 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1921 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1981 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2041 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2101 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2161 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2221 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2281 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2341 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     2401 cgaggaagct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     2461 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     2521 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     2581 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     2641 gctgcggtaa agctcatcag cgtggtcgtg cagcgattca cagatgtctg cctgttcatc
     2701 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     2761 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     2821 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     2881 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     2941 gcgggaccag agaaaaatca ctcagggtca atgccagccg aacgccagca agacgtagcc
     3001 cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     3061 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     3121 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     3181 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     3241 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     3301 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     3361 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3421 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     3481 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     3541 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     3601 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     3661 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     3721 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     3781 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     3841 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     3901 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     3961 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     4021 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     4081 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     4141 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     4201 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     4261 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     4321 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     4381 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     4441 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     4501 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     4561 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg ccggcatgcc
     4621 gccctttcgt cttcaagaat taattcccaa ttccccaggc atcaaataaa acgaaaggct
     4681 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     4741 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     4801 gcaggacgcc cgccataaac tgccaggaat taattcccca ggcatcaaat aaaacgaaag
     4861 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     4921 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     4981 cgggcaggac gcccgccata aactgccagg aattggggat cggaattaat tcccggttta
     5041 aaccggggat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     5101 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acttatgaaa
     5161 atcgaagaag gtaaactggt aatcggttcc ctggagggtt gctttgccaa gggtaccaat
     5221 gttttaatgg cggatgggtc tattgaatgt attgaaaaca ttgaggttgg taataaggtc
     5281 atgggtaaag atggcagacc tcgtgaggta attaaattgc ccagaggaag agaaactatg
     5341 tacagcgtcg tgcagaaaag tcagcacaga gcccacaaaa gtgactcaag tcgtgaagtg
     5401 ccagaattac tcaagtttac gtgtaatgcg acccatgagt tggttgttag aacacctcgt
     5461 agtgtccgcc gtttgtctcg taccattaag ggtgtcgaat attttgaagt tattactttt
     5521 gagatgggcc aaaagaaagc ccccgacggt agaattgttg agcttgtcaa ggaagtttca
     5581 aagagctacc caatatctga ggggcctgag agagccaacg aattagtaga atcctataga
     5641 aaggcttcaa ataaagctta ttttgagtgg actattgagg ccagagatct ttctctgttg
     5701 ggttcccatg ttcgtaaagc tacctaccag acttacgctc caattcttta tgagaatgac
     5761 cactttttcg actacatgca aaaaagtaag tttcatctca ccattgaagg tccaaaagta
     5821 cttgcttatt tacttggttt atggattggt gatggattgt ctgacagggc aactttttcg
     5881 gttgattcca gagatacttc tttgatggaa cgtgttactg aatatgctga aaagttgaat
     5941 ttgtgcgccg agtataagga cagaaaagaa ccacaagttg ccaaaactgt taatttgtac
     6001 tctaaagttg tcagaggtgc tagcacaaat cctggtgtat ccgcttggca ggtcaacaca
     6061 gcttatactg cgggacaatt ggtcacatat aacggcaaga cgtataaatg tttgcagccc
     6121 cacacctcct tggcaggatg ggaaccatcc aacgttcctg ccttgtggca gcttcaaggt
     6181 ggccacggtg gtattcgcaa taatcttaat actgagaatc cattatggga cgctattgtt
     6241 ggcttaggat tcttgaagga cggtgtcaaa aatattcctt ctttcttgtc tacggacaat
     6301 atcggtactc gtgaaacatt tcttgctggt ctaattgatt ctgatggcta tgttactgat
     6361 gagcatggta ttaaagcaac aataaagaca attcatactt ctgtcagaga tggtttggtt
     6421 tcccttgctc gttctttagg cttagtagtc tcggttaacg cagaacctgc taaggttgac
     6481 atgaatgtca ccaaacataa aattagttat gctatttata tgtctggtgg agatgttttg
     6541 cttaacgttc tttcgaagtg tgccggctct aaaaaattca ggcctgctcc cgccgctgct
     6601 tttgcacgtg agtgccgcgg attttatttc gagttacaag aattgaagga agacgattat
     6661 tatgggatta ctttatctga tgattctgat catcagtttt tgcttggatc tcaggttgtt
     6721 gtacagaaca tggacccgcg tgaatgtgtc tgtatgagtg gtggtatttg tatgtgtggt
     6781 gataactgta agtgtacgac gtgtaactgt aaaacgtatt ggaaaagttg ctgtccgtgc
     6841 tgtccgccgg gttgcgcaaa gtgtgcccgt ggttgcattt gtaaaggcgg tagcgataag
     6901 tgctcttgct gtccgtaact gcaggtaatt aaataactag ttgatccggc tgctaacaaa
     6961 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     7021 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggataa
     7081 ctacgtcagg tggcacttt
//
