LOCUS       sequence-203322-        7096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 1f.
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: MT1F
FEATURES             Location/Qualifiers
     source          1..7096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        19..123
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(124..192,193..984)
                     /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(342..361)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1026..1539)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1221..1242)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1433..1452
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1650..2238
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2139..2158
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2392..2409
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2431..2450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2591..2613
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2607..2798)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3355..4437)
                     /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     4399..4418
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4438..4515)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4668..4754
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4851..4937
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5072..5091
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5072..5090
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5091..5115
                     /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             5130..5152
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5205..6023
                     /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             6030..6182
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6186..6734
                     /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(6994..7012)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7008..7055
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
       61 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
      121 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
      181 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
      241 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
      301 cccgaagaac gtttcccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
      361 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
      421 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
      481 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
      541 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
      601 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
      661 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
      721 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
      781 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
      841 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
      901 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
      961 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     1021 gatttacccc ggttgataat cagaaaagcc ccaaaaacag gaagattgta taagcaaata
     1081 tttaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     1141 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagcccg
     1201 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     1261 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     1321 ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     1381 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     1441 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     1501 ccacacccgc cgcgcttaat gcgccgctac agggcgcgta aaaggatcta ggtgaagatc
     1561 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1621 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1681 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1741 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     1801 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1861 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1921 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     1981 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2041 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2101 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2161 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2221 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2281 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2341 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2401 gtgagcgagg aagctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2461 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     2521 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     2581 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     2641 aggcagctgc ggtaaagctc atcagcgtgg tcgtgcagcg attcacagat gtctgcctgt
     2701 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     2761 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     2821 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     2881 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     2941 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agccgaacgc cagcaagacg
     3001 tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     3061 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     3121 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     3181 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     3241 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     3301 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     3361 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3421 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     3481 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     3541 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     3601 gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga
     3661 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     3721 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     3781 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     3841 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     3901 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     3961 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     4021 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     4081 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     4141 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     4201 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     4261 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     4321 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     4381 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     4441 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     4501 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     4561 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgccggc
     4621 atgccgccct ttcgtcttca agaattaatt cccaattccc caggcatcaa ataaaacgaa
     4681 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     4741 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     4801 ggcgggcagg acgcccgcca taaactgcca ggaattaatt ccccaggcat caaataaaac
     4861 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     4921 tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag
     4981 ggtggcgggc aggacgcccg ccataaactg ccaggaattg gggatcggaa ttaattcccg
     5041 gtttaaaccg gggatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     5101 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatactta
     5161 tgaaaatcga agaaggtaaa ctggtaatcg gttccctgga gggttgcttt gccaagggta
     5221 ccaatgtttt aatggcggat gggtctattg aatgtattga aaacattgag gttggtaata
     5281 aggtcatggg taaagatggc agacctcgtg aggtaattaa attgcccaga ggaagagaaa
     5341 ctatgtacag cgtcgtgcag aaaagtcagc acagagccca caaaagtgac tcaagtcgtg
     5401 aagtgccaga attactcaag tttacgtgta atgcgaccca tgagttggtt gttagaacac
     5461 ctcgtagtgt ccgccgtttg tctcgtacca ttaagggtgt cgaatatttt gaagttatta
     5521 cttttgagat gggccaaaag aaagcccccg acggtagaat tgttgagctt gtcaaggaag
     5581 tttcaaagag ctacccaata tctgaggggc ctgagagagc caacgaatta gtagaatcct
     5641 atagaaaggc ttcaaataaa gcttattttg agtggactat tgaggccaga gatctttctc
     5701 tgttgggttc ccatgttcgt aaagctacct accagactta cgctccaatt ctttatgaga
     5761 atgaccactt tttcgactac atgcaaaaaa gtaagtttca tctcaccatt gaaggtccaa
     5821 aagtacttgc ttatttactt ggtttatgga ttggtgatgg attgtctgac agggcaactt
     5881 tttcggttga ttccagagat acttctttga tggaacgtgt tactgaatat gctgaaaagt
     5941 tgaatttgtg cgccgagtat aaggacagaa aagaaccaca agttgccaaa actgttaatt
     6001 tgtactctaa agttgtcaga ggtgctagca caaatcctgg tgtatccgct tggcaggtca
     6061 acacagctta tactgcggga caattggtca catataacgg caagacgtat aaatgtttgc
     6121 agccccacac ctccttggca ggatgggaac catccaacgt tcctgccttg tggcagcttc
     6181 aaggtggcca cggtggtatt cgcaataatc ttaatactga gaatccatta tgggacgcta
     6241 ttgttggctt aggattcttg aaggacggtg tcaaaaatat tccttctttc ttgtctacgg
     6301 acaatatcgg tactcgtgaa acatttcttg ctggtctaat tgattctgat ggctatgtta
     6361 ctgatgagca tggtattaaa gcaacaataa agacaattca tacttctgtc agagatggtt
     6421 tggtttccct tgctcgttct ttaggcttag tagtctcggt taacgcagaa cctgctaagg
     6481 ttgacatgaa tgtcaccaaa cataaaatta gttatgctat ttatatgtct ggtggagatg
     6541 ttttgcttaa cgttctttcg aagtgtgccg gctctaaaaa attcaggcct gctcccgccg
     6601 ctgcttttgc acgtgagtgc cgcggatttt atttcgagtt acaagaattg aaggaagacg
     6661 attattatgg gattacttta tctgatgatt ctgatcatca gtttttgctt ggatctcagg
     6721 ttgttgtaca gaacatggac ccgaattgtt catgtgcggc aggcgtctcg tgtacgtgtg
     6781 caggctcgtg taagtgtaaa gaatgtaaat gtacctcgtg taaaaagagc tgctgttctt
     6841 gctgtccggt gggttgcagc aaatgtgcgc agggctgcgt ttgtaaaggt gccagtgaaa
     6901 agtgctcctg ctgtgattaa ctgcaggtaa ttaaataact agttgatccg gctgctaaca
     6961 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     7021 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     7081 aactacgtca ggtggc
//
