LOCUS       sequence-430159-        5540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a human zinc 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 5540)
  TITLE     Shanbhag lab plasmids
REFERENCE   2  (bases 1 to 5540)
  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-rHMT
FEATURES             Location/Qualifiers
     source          1..5540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1076..1267
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1261..1283)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1369..1508
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1424..1443
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1523..1540)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1694..2282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1774..1793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2393..2412)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2404..3219
                     /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             2404..3219
                     /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(2477..2496)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3311..3766)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3448..3469)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3660..3679
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3803..3850)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3846..3864
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3917..3934)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4166..4183)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(4199..4243)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     RBS             4254..4276
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4291..4315
                     /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(4315..4334)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4316..4334)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4422..4441)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4647..4724
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4725..5540,1..267)
                     /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(4744..4763)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
       61 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
      121 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
      181 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      241 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
      301 ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg
      361 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
      421 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
      481 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
      541 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
      601 ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
      661 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
      721 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
      781 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
      841 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
      901 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
      961 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
     1021 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
     1081 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
     1141 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
     1201 cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa
     1261 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     1321 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     1381 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     1441 gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     1501 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1561 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1621 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1681 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1741 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     1801 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     1861 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     1921 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1981 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2041 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2101 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     2161 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2221 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2281 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2341 aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     2401 gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg
     2461 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     2521 atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     2581 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     2641 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     2701 gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     2761 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     2821 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     2881 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     2941 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     3001 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     3061 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     3121 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     3181 aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata
     3241 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3301 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     3361 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     3421 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     3481 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     3541 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     3601 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     3661 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     3721 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc
     3781 cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg
     3841 gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt cgggctttgt
     3901 tagcagccgg atctcagtgg tggtggtggt ggtgctcgag ttattacgcc gccgccgcct
     3961 tcttacaaca gctgcatttt tcgctcgcac ccttgcacac gcaaccctgc gcgcatttcg
     4021 cgcagctcat cgggcagcag ctgcagcacg ccgccgccgc cttcttgcag ctgttgcact
     4081 tgcactcttt gcacttgcag ctaccggtgc aggtgcagct gccaccggtc gcgcagctgc
     4141 acgccgccgc agctttccag cccatggaac cgcgtggcac cagggtaccc agatctgggc
     4201 tgtccatgtg ctggcgttcg aatttagcag cagcggtttc tttcatatgt atatctcctt
     4261 cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat
     4321 agtgagtcgt attaatttcg cgggatcgag atcgatctcg atcctctacg ccggacgcat
     4381 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
     4441 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
     4501 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
     4561 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
     4621 gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta
     4681 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4741 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     4801 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4861 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4921 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     4981 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5041 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5101 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5161 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     5221 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5281 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5341 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5401 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5461 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5521 cggacatctc ggtagtggga
//
