LOCUS       sequence-203324-        6916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 1g.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6916)
  TITLE     Krezel Lab Plasmids
REFERENCE   2  (bases 1 to 6916)
  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
FEATURES             Location/Qualifiers
     source          1..6916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      115..201
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     336..355
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        336..354
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    355..379
                     /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             394..416
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             469..1287
                     /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             1294..1446
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             1450..1998
                     /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(2261..2279)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2275..2322
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2382..2486
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2487..2555,2556..3347)
                     /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(2705..2724)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3389..3902)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3584..3605)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3796..3815
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4013..4601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4502..4521
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4755..4772
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4794..4813)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4954..4976
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4970..5161)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5718..6800)
                     /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
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMAERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6762..6781
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6801..6878)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
       61 tgccggcatg ccgccctttc gtcttcaaga attaattccc aattccccag gcatcaaata
      121 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
      181 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
      241 ggagggtggc gggcaggacg cccgccataa actgccagga attggggatc ggaattaatt
      301 cccggtttaa accggggatc tcgatcccgc gaaattaata cgactcacta taggggaatt
      361 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
      421 cttatgaaaa tcgaagaagg taaactggta atcggttccc tggagggttg ctttgccaag
      481 ggtaccaatg ttttaatggc ggatgggtct attgaatgta ttgaaaacat tgaggttggt
      541 aataaggtca tgggtaaaga tggcagacct cgtgaggtaa ttaaattgcc cagaggaaga
      601 gaaactatgt acagcgtcgt gcagaaaagt cagcacagag cccacaaaag tgactcaagt
      661 cgtgaagtgc cagaattact caagtttacg tgtaatgcga cccatgagtt ggttgttaga
      721 acacctcgta gtgtccgccg tttgtctcgt accattaagg gtgtcgaata ttttgaagtt
      781 attacttttg agatgggcca aaagaaagcc cccgacggta gaattgttga gcttgtcaag
      841 gaagtttcaa agagctaccc aatatctgag gggcctgaga gagccaacga attagtagaa
      901 tcctatagaa aggcttcaaa taaagcttat tttgagtgga ctattgaggc cagagatctt
      961 tctctgttgg gttcccatgt tcgtaaagct acctaccaga cttacgctcc aattctttat
     1021 gagaatgacc actttttcga ctacatgcaa aaaagtaagt ttcatctcac cattgaaggt
     1081 ccaaaagtac ttgcttattt acttggttta tggattggtg atggattgtc tgacagggca
     1141 actttttcgg ttgattccag agatacttct ttgatggaac gtgttactga atatgctgaa
     1201 aagttgaatt tgtgcgccga gtataaggac agaaaagaac cacaagttgc caaaactgtt
     1261 aatttgtact ctaaagttgt cagaggtgct agcacaaatc ctggtgtatc cgcttggcag
     1321 gtcaacacag cttatactgc gggacaattg gtcacatata acggcaagac gtataaatgt
     1381 ttgcagcccc acacctcctt ggcaggatgg gaaccatcca acgttcctgc cttgtggcag
     1441 cttcaaggtg gccacggtgg tattcgcaat aatcttaata ctgagaatcc attatgggac
     1501 gctattgttg gcttaggatt cttgaaggac ggtgtcaaaa atattccttc tttcttgtct
     1561 acggacaata tcggtactcg tgaaacattt cttgctggtc taattgattc tgatggctat
     1621 gttactgatg agcatggtat taaagcaaca ataaagacaa ttcatacttc tgtcagagat
     1681 ggtttggttt cccttgctcg ttctttaggc ttagtagtct cggttaacgc agaacctgct
     1741 aaggttgaca tgaatgtcac caaacataaa attagttatg ctatttatat gtctggtgga
     1801 gatgttttgc ttaacgttct ttcgaagtgt gccggctcta aaaaattcag gcctgctccc
     1861 gccgctgctt ttgcacgtga gtgccgcgga ttttatttcg agttacaaga attgaaggaa
     1921 gacgattatt atgggattac tttatctgat gattctgatc atcagttttt gcttggatct
     1981 caggttgttg tacagaacat ggacccgaat tgttcctgtg ccgcggctgg cgtgtcatgt
     2041 acctgtgcgt cgtcgtgtaa gtgtaaagaa tgtaaatgta cctcgtgtaa aaagagctgc
     2101 tgttcttgct gtccggtggg ttgcgcaaaa tgtgcacagg gttgcatttg taaaggtgca
     2161 agtgaaaagt gcagctgctg tgcgtaactg caggtaatta aataactagt tgatccggct
     2221 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     2281 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     2341 tccggataac tacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2401 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2461 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2521 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2581 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2641 gtaagatcct tgagagtttt cgccccgaag aacgtttccc aatgatgagc acttttaaag
     2701 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     2761 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2821 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2881 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2941 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3001 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3061 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3121 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3181 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3241 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3301 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3361 tttactcata tatactttag attgatttac cccggttgat aatcagaaaa gccccaaaaa
     3421 caggaagatt gtataagcaa atatttaaat tgtaaacgtt aatattttgt taaaattcgc
     3481 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3541 ttataaatca aaagaatagc ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3601 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3661 tggcccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt gccgtaaagc
     3721 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3781 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3841 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3901 gtaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3961 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4021 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4081 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4141 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4201 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4261 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4321 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4381 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4441 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4501 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4561 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4621 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4681 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4741 gaacgaccga gcgcagcgag tcagtgagcg aggaagctat ggtgcactct cagtacaatc
     4801 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     4861 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4921 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4981 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgca
     5041 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     5101 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     5161 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     5221 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     5281 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     5341 gccagccgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg
     5401 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     5461 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     5521 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     5581 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     5641 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     5701 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5761 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     5821 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     5881 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     5941 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
     6001 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     6061 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     6121 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     6181 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     6241 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     6301 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     6361 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     6421 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     6481 caggcttcga cgccgcttcg ttctgccatc gacaccacca cgctggcacc cagttgatcg
     6541 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     6601 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     6661 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     6721 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     6781 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6841 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     6901 atgcgactcc tgcatt
//
