LOCUS       sequence-203325-        7096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 1g subisoform 2 with deletion of Alanine 10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7096)
  TITLE     Krezel Lab Plasmids
REFERENCE   2  (bases 1 to 7096)
  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: MT1G_isoform2
FEATURES             Location/Qualifiers
     source          1..7096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     150..169
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        150..168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    169..193
                     /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             208..230
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             283..1101
                     /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             1108..1260
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             1264..1812
                     /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(2072..2090)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2086..2133
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2193..2297
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2298..2366,2367..3158)
                     /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(2516..2535)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3200..3713)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3395..3416)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3607..3626
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      3824..4412
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4313..4332
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4566..4583
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4605..4624)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4765..4787
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4781..4972)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5529..6611)
                     /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     6573..6592
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6612..6689)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      6842..6928
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(7025..7096,1..15)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
       61 tggcgggcag gacgcccgcc ataaactgcc aggaattggg gatcggaatt aattcccggt
      121 ttaaaccggg gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
      181 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacttatg
      241 aaaatcgaag aaggtaaact ggtaatcggt tccctggagg gttgctttgc caagggtacc
      301 aatgttttaa tggcggatgg gtctattgaa tgtattgaaa acattgaggt tggtaataag
      361 gtcatgggta aagatggcag acctcgtgag gtaattaaat tgcccagagg aagagaaact
      421 atgtacagcg tcgtgcagaa aagtcagcac agagcccaca aaagtgactc aagtcgtgaa
      481 gtgccagaat tactcaagtt tacgtgtaat gcgacccatg agttggttgt tagaacacct
      541 cgtagtgtcc gccgtttgtc tcgtaccatt aagggtgtcg aatattttga agttattact
      601 tttgagatgg gccaaaagaa agcccccgac ggtagaattg ttgagcttgt caaggaagtt
      661 tcaaagagct acccaatatc tgaggggcct gagagagcca acgaattagt agaatcctat
      721 agaaaggctt caaataaagc ttattttgag tggactattg aggccagaga tctttctctg
      781 ttgggttccc atgttcgtaa agctacctac cagacttacg ctccaattct ttatgagaat
      841 gaccactttt tcgactacat gcaaaaaagt aagtttcatc tcaccattga aggtccaaaa
      901 gtacttgctt atttacttgg tttatggatt ggtgatggat tgtctgacag ggcaactttt
      961 tcggttgatt ccagagatac ttctttgatg gaacgtgtta ctgaatatgc tgaaaagttg
     1021 aatttgtgcg ccgagtataa ggacagaaaa gaaccacaag ttgccaaaac tgttaatttg
     1081 tactctaaag ttgtcagagg tgctagcaca aatcctggtg tatccgcttg gcaggtcaac
     1141 acagcttata ctgcgggaca attggtcaca tataacggca agacgtataa atgtttgcag
     1201 ccccacacct ccttggcagg atgggaacca tccaacgttc ctgccttgtg gcagcttcaa
     1261 ggtggccacg gtggtattcg caataatctt aatactgaga atccattatg ggacgctatt
     1321 gttggcttag gattcttgaa ggacggtgtc aaaaatattc cttctttctt gtctacggac
     1381 aatatcggta ctcgtgaaac atttcttgct ggtctaattg attctgatgg ctatgttact
     1441 gatgagcatg gtattaaagc aacaataaag acaattcata cttctgtcag agatggtttg
     1501 gtttcccttg ctcgttcttt aggcttagta gtctcggtta acgcagaacc tgctaaggtt
     1561 gacatgaatg tcaccaaaca taaaattagt tatgctattt atatgtctgg tggagatgtt
     1621 ttgcttaacg ttctttcgaa gtgtgccggc tctaaaaaat tcaggcctgc tcccgccgct
     1681 gcttttgcac gtgagtgccg cggattttat ttcgagttac aagaattgaa ggaagacgat
     1741 tattatggga ttactttatc tgatgattct gatcatcagt ttttgcttgg atctcaggtt
     1801 gttgtacaga acatggaccc gaattgttcc tgtgcggctg gcgtgtcatg tacctgtgcg
     1861 tcgtcgtgta agtgtaaaga atgtaaatgt acctcgtgta aaaagagctg ctgttcttgc
     1921 tgtccggtgg gttgcgcaaa atgtgcacag ggttgcattt gtaaaggtgc aagtgaaaag
     1981 tgcagctgct gtgcgtaact gcaggtaatt aaataactag ttgatccggc tgctaacaaa
     2041 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2101 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggataa
     2161 ctacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2221 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2281 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2341 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2401 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2461 ttgagagttt tcgccccgaa gaacgtttcc caatgatgag cacttttaaa gttctgctat
     2521 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     2581 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2641 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2701 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2761 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2821 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2881 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2941 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3001 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3061 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3121 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3181 atatacttta gattgattta ccccggttga taatcagaaa agccccaaaa acaggaagat
     3241 tgtataagca aatatttaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt
     3301 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     3361 aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     3421 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     3481 acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     3541 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     3601 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     3661 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtaaaagga
     3721 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3781 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3841 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3901 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3961 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4021 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4081 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4141 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4201 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4261 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4321 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4381 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4441 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4501 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4561 agcgcagcga gtcagtgagc gaggaagcta tggtgcactc tcagtacaat ctgctctgat
     4621 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4681 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4741 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4801 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac
     4861 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4921 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4981 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     5041 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     5101 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagccga
     5161 acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct
     5221 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     5281 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     5341 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     5401 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     5461 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     5521 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5581 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     5641 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     5701 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     5761 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
     5821 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     5881 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     5941 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     6001 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     6061 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     6121 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     6181 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     6241 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     6301 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     6361 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     6421 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     6481 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     6541 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     6601 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     6661 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     6721 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     6781 tggtgcatgc cggcatgccg ccctttcgtc ttcaagaatt aattcccaat tccccaggca
     6841 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     6901 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca
     6961 acggcccgga gggtggcggg caggacgccc gccataaact gccaggaatt aattccccag
     7021 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     7081 gtcggtgaac gctctc
//
