LOCUS       sequence-430152-        5549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a proteobacteria metallothionein fused with the S-tag at the
            N-terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5549)
  TITLE     Shanbhag lab plasmids
REFERENCE   2  (bases 1 to 5549)
  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: Stag-ComMT
FEATURES             Location/Qualifiers
     source          1..5549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(63..82)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(682..701)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             693..1508
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             693..1508
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(766..785)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1600..2055)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1737..1758)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1949..1968
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2092..2139)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2135..2153
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2206..2223)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2464..2481)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2497..2541)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     RBS             2552..2574
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2589..2613
                     /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)."
     primer_bind     complement(2613..2632)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2614..2632)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2720..2739)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2945..3022
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3023..4105
                     /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     complement(3042..3061)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4914..5105
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5099..5121)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5207..5346
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5262..5281
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5361..5378)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5532..5549,1..571))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
       61 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
      121 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
      181 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
      241 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      301 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
      361 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
      421 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
      481 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
      541 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
      601 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca ataaaactgt
      661 ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt
      721 gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
      781 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc
      841 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
      901 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
      961 ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat
     1021 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     1081 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg
     1141 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
     1201 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac
     1261 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     1321 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     1381 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     1441 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     1501 ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     1561 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     1621 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     1681 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     1741 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     1801 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     1861 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     1921 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     1981 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2041 cgctacaggg cgcgtcccat tcgccaatcc ggatatagtt cctcctttca gcaaaaaacc
     2101 cctcaagacc cgtttagagg ccccaagggg ttatgctagt tattgctcag cggtggcagc
     2161 agccaactca gcttcctttc gggctttgtt agcagccgga tctcagtggt ggtggtggtg
     2221 gtgctcgagt tattacgggc tgatcggatc gctcgccggg aaggtctcat ccagcgcgtt
     2281 gtccaccgcg ctttcacccg gtttctcgcc gcagtggcag tcctgcatac ggcacggttc
     2341 gcccttacgg tgaccgctcg cgcacgcctc gcagcaatac gctttgccat cacgttgcag
     2401 cgccttcgca accacggtgc agctgcagtg ggtgcacgcg caacgctggt cgtacatgcc
     2461 catggaaccg cgtggcacca gggtacccag atctgggctg tccatgtgct ggcgttcgaa
     2521 tttagcagca gcggtttctt tcatatgtat atctccttct taaagttaaa caaaattatt
     2581 tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
     2641 ggatcgagat cgatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
     2701 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
     2761 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
     2821 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
     2881 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat
     2941 cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     3001 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     3061 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     3121 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     3181 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     3241 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     3301 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     3361 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     3421 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     3481 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     3541 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     3601 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     3661 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     3721 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     3781 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gacatctcgg tagtgggata
     3841 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     3901 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     3961 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     4021 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     4081 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     4141 aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     4201 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     4261 taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg
     4321 cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct
     4381 tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg
     4441 cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg
     4501 gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct
     4561 gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt
     4621 aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag
     4681 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac
     4741 cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac
     4801 gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt
     4861 tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca
     4921 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc
     4981 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg
     5041 accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     5101 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     5161 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     5221 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     5281 actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     5341 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5401 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5461 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5521 ttgctggcgt ttttccatag gctccgccc
//
