LOCUS       sequence-430151-        5510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a yeast copper 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 5510)
  TITLE     Shanbhag lab plasmids
REFERENCE   2  (bases 1 to 5510)
  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-YCup1
FEATURES             Location/Qualifiers
     source          1..5510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             643..834
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(828..850)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    936..1075
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     991..1010
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1090..1107)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1261..1849)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1341..1360)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1960..1979)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1971..2786
                     /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             1971..2786
                     /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(2044..2063)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2878..3333)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3015..3036)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3227..3246
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3370..3417)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3413..3431
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3484..3501)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3703..3720)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(3736..3780)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     RBS             3791..3813
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3828..3852
                     /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(3852..3871)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3853..3871)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3959..3978)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4184..4261
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4262..5344
                     /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(4281..4300)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc
       61 ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt
      121 cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg
      181 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
      241 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
      301 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
      361 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
      421 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
      481 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
      541 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
      601 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
      661 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
      721 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
      781 gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
      841 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
      901 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
      961 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     1021 accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
     1081 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     1141 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1201 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1261 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1321 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1381 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1441 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1501 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1561 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1621 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1681 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     1741 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     1801 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1861 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1921 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     1981 ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat
     2041 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt
     2101 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     2161 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     2221 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggga
     2281 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     2341 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     2401 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     2461 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     2521 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     2581 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     2641 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     2701 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     2761 atttgatgct cgatgagttt ttctaagaat taattcatga gcggatacat atttgaatgt
     2821 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     2881 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     2941 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     3001 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     3061 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     3121 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     3181 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     3241 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     3301 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccaatccgg atatagttcc
     3361 tcctttcagc aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt atgctagtta
     3421 ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg gctttgttag cagccggatc
     3481 tcagtggtgg tggtggtggt gctcgagtta ttacttaccg ctgcagcagc ttttcttggt
     3541 ttcctcgctt ttgttgccgc acgggcactt atcgtcgctg ttgcaaccgg tcgggcagct
     3601 gcagctcttc tggcactgtt cgttgttctt gcagctgccg cattggcact ggcattcgtg
     3661 accctcgttc tgaaagttga tcagctcgct gaaccagccc atggaaccgc gtggcaccag
     3721 ggtacccaga tctgggctgt ccatgtgctg gcgttcgaat ttagcagcag cggtttcttt
     3781 catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     3841 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc
     3901 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
     3961 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
     4021 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
     4081 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
     4141 ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg
     4201 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     4261 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     4321 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     4381 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     4441 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     4501 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     4561 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     4621 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     4681 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     4741 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     4801 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     4861 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     4921 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     4981 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     5041 cgggctgcgc gttggtgcgg acatctcggt agtgggatac gacgataccg aagacagctc
     5101 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     5161 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     5221 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     5281 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     5341 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga
     5401 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     5461 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
//
