LOCUS       sequence-8215-ex        5477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tandem-affinity purification plasmid; streptavidin-(streptag) and 
            calmodulin-(CBP) binding affinity tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5477)
  AUTHORS   Angers S, Thorpe CJ, Biechele TL, Goldenberg SJ, Zheng N, MacCoss 
            MJ, Moon RT
  TITLE     The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the 
            Wnt-beta-catenin pathway by targeting Dishevelled for degradation.
  JOURNAL   Nat Cell Biol. 2006 Apr . 8(4):348-57.
  PUBMED    16547521
REFERENCE   2  (bases 1 to 5477)
  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"; journalName: "Nat Cell 
            Biol. 2006 Apr . 8(4):348-57."
COMMENT     Sequence Label: V51 pIRESpuro-GLUE (pGLUE)
FEATURES             Location/Qualifiers
     source          1..5477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..612
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        613..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     767..787
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     861..880
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        861..879
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      921..930
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             927..1040
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             1059..1079
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1086..1112
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1086..1112
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1113..1190
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     intron          1336..1565
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1548..1567
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1623..2197,2198..2200,2201..2209)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1790..1807)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2017..2036
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2229..2828
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     primer_bind     complement(2229..2248)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2725..2745
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2964..2981)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2970..3194
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3241..3260
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3320..3336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3320..3336)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3333..3355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3344..3360
                     /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)."
     promoter        complement(join(3368..3374,3375..3392,3393..3398))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3413..3434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3551..3568)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3722..4310)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3802..3821)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4481..5272,5273..5341))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5104..5123
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5342..5446)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccacgacccc
      361 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      421 tgacgtcaat gggtggacta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      481 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      541 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      601 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      661 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      721 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      781 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact
      841 gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc ttggtaccga
      901 gctcggatcg atttaattaa gccaccatgg acgagaagac caccggctgg cggggcggcc
      961 acgtggtgga gggcctggcc ggcgagctgg agcagctgcg ggcccggctg gagcaccacc
     1021 cccagggcca gcgggagccc ggcggaagcc ccggtggcga gaacctgtac ttccagggcg
     1081 caagctaccc ctacgacgtg cccgactacg ccaagcggcg gtggaagaag aacttcatcg
     1141 ccgtgagcgc cgccaaccgg ttcaagaaga tcagcagcag cggcgccctg attgatgggg
     1201 gatctggccc cggcggcggc gcgcctgtac agatatctgc ggccgcgtcg acggaattcg
     1261 cccttttcgg atccactagt aacggccgcc agtgtgctgg aattaattcg ctgtctgcga
     1321 gggccagctg ttggggtgag tactccctct caaaagcggg catgacttct gcgctaagat
     1381 tgtcagtttc caaaaacgag gaggatttga tattcacctg gcccgcggtg atgcctttga
     1441 gggtggccgc gtccatctgg tcagaaaaga caatcttttt gttgtcaagc ttgaggtgtg
     1501 gcaggcttga gatctggcca tacacttgag tgacaatgac atccactttg cctttctctc
     1561 cacaggtgtc cactcccagg tccaactgca ggtcgagcat gcatctaggg cggccaattc
     1621 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg
     1681 tgtgcgtttg tctatatgtg attttccacc atattgccgt cttttggcaa tgtgagggcc
     1741 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa
     1801 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
     1861 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
     1921 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
     1981 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
     2041 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
     2101 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
     2161 ggggacgtgg ttttcctttg aaaaacacga tgataagctt gccacaaccc acaaggagac
     2221 gaccttccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
     2281 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     2341 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     2401 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     2461 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     2521 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     2581 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     2641 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     2701 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     2761 tcaccgccga cgtcgagtgc ccgaaggacc gcgcgacctg gtgcatgacc cgcaagcccg
     2821 gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg
     2881 gctccgaccg aagccgaccc gggcggcccc gccgaccccg cacccgcccc cgaggcccac
     2941 cgactctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3001 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3061 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3121 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3181 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgagtg cattctagtt
     3241 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     3301 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3361 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3421 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3481 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3541 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3601 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3661 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3721 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3781 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3841 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3901 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3961 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4021 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4081 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4141 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4201 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4261 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4321 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4381 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4441 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4501 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4561 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4621 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4681 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4741 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4801 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4861 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4921 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4981 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5041 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5101 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5161 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5221 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5281 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5341 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5401 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5461 aagtgccacc tgacgtc
//
