LOCUS       sequence-8217-ex        7639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7639)
  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 7639)
  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: M77 pIRESpuro-GLUE-hDsh3
FEATURES             Location/Qualifiers
     source          1..7639
                     /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..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      923..932
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             929..1042
                     /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             1061..1081
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1088..1114
                     /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             1088..1114
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1115..1192
                     /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"
     protein_bind    1841..1852
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     intron          3498..3727
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     3710..3729
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(3785..4359,4360..4362,4363..4371)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3952..3969)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4179..4198
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4391..4990
                     /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(4391..4410)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4887..4907
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5126..5143)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5132..5356
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5403..5422
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5482..5498)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5482..5498)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5495..5517)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5506..5522
                     /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(5530..5536,5537..5554,5555..5560))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5575..5596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5713..5730)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5884..6472)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5964..5983)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6643..7434,7435..7503))
                     /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     7266..7285
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7504..7608)
                     /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 tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgatttaatt aagccaccat ggacgagaag accaccggct ggcggggcgg
      961 ccacgtggtg gagggcctgg ccggcgagct ggagcagctg cgggcccggc tggagcacca
     1021 cccccagggc cagcgggagc ccggcggaag ccccggtggc gagaacctgt acttccaggg
     1081 cgcaagctac ccctacgacg tgcccgacta cgccaagcgg cggtggaaga agaacttcat
     1141 cgccgtgagc gccgccaacc ggttcaagaa gatcagcagc agcggcgccc tgattgatgg
     1201 gggatctggc cccggcggcg gcgcgcctgt acagattcga gagggtggaa gtggaccagg
     1261 tggtatgggc gagaccaaga tcatctacca cttggatggg caggagacgc cgtaccttgt
     1321 gaagctgccc ctgcccgccg agcgcgtcac cttggcggac tttaagggcg ttttgcagcg
     1381 acccagctat aagttcttct tcaagtctat ggacgacgat ttcggagtgg tgaaggagga
     1441 gatctcggat gacaatgcca agctaccatg cttcaatggc cgggtggtgt cctggctggt
     1501 gtcagctgag ggctcacacc cagacccagc ccccttctgt gctgataacc catcggagct
     1561 gccaccacct atggagcgca cgggaggcat cggggactcc cgacccccat ccttccaccc
     1621 tcatgctggt gggggcagcc aggagaacct ggacaatgac acagagacgg actctttggt
     1681 gtctgcccag cgagagcggc cacgccggag ggatggccca gagcatgcaa cccggctaaa
     1741 tggaactgcg aagggggaac ggcggcgaga accagggggt tatgatagct catccaccct
     1801 tatgagcagt gagctggaga ccaccagctt ctttgactca gatgaggatg actccaccag
     1861 caggttcagc agctccacag aacagagcag tgcctcacgc ctgatgagaa gacacaagcg
     1921 gcggcggcgg aagcagaagg tttctcggat tgagcggtcc tcgtccttca gcagcatcac
     1981 ggactccacc atgtcactca acatcatcac ggtcactctc aacatggaaa aatataactt
     2041 cttgggcatc tccattgtgg gccaaagcaa cgagcgtggt gacggcggca tctacattgg
     2101 ctctatcatg aagggtgggg ccgtggctgc tgatggacgc atcgagccag gagatatgtt
     2161 gttacaggta aacgagatca actttgagaa catgagtaat gacgatgcag tccgggtact
     2221 gcgggagatt gtgcacaaac cggggcccat caccctgact gtagccaagt gctgggaccc
     2281 aagtccacgt ggttgcttca cattgcccag gagcgagccc atccggccca ttgaccctgc
     2341 ggcctgggtc tcccacactg cagccatgac cggcaccttc cctgcatacg gcatgagccc
     2401 ctccctgagc accatcacct ccaccagctc ctccatcacc agttccatcc ctgacacaga
     2461 gcgcctagac gacttccact tgtccatcca cagtgacatg gctgccatcg taaaagccat
     2521 ggcctcccct gaatcagggt tggaggtccg tgaccgcatg tggctcaaga ttaccatccc
     2581 taatgctttc atcggctcag atgtggtgga ctggctgtac cacaatgtgg aaggcttcac
     2641 ggaccggagg gaggcccgca agtatgccag caacctgctg aaagctggct tcatccgcca
     2701 taccgtcaac aagatcacct tctccgagca gtgctactac atcttcggtg acctctgcgg
     2761 caacatggcc aacctgtctc tccacgatca cgatggctcc agtggcgcct ctgaccagga
     2821 cacactggcc cctttgccgc acccgggggc cgccccttgg cccatggctt tcccgtacca
     2881 gtacccgcca cgcccgcacc catacaaccc gcacccgggc ttgggggagc tgggctacag
     2941 ctacggcggg ggcagcgcca gcagtcagca cagcgaaggc agtcggagca gtggctccaa
     3001 ccgtagcggc agcgatcgga ggaaggagaa ggacccgaag gccggggact ccaagtccgg
     3061 gggcagcggc agcgaatcgg accacaccac acgcagcagc ctgcgggggc cgcgggagcg
     3121 ggcgcccagc gagcgctcag ggccggcggc cagcgagcac agccaccgca gccaccattc
     3181 cctggccagc agccttcgca gccaccacac acacccgagc tacggtcctc ccggagtgcc
     3241 ccctctctac ggccccccca tgctgatgat gcccccgccg cccgcggcca tggggccccc
     3301 aggagcccct ccgggccgcg acctggcctc agtgcccccg gaactgaccg ccagcagaca
     3361 gtccttccgc atggccatgg gaaaccccag tgagttcttt gtggatgtga tgtgagaatt
     3421 cgcccttttc ggatccacta gtaacggccg ccagtgtgct ggaattaatt cgctgtctgc
     3481 gagggccagc tgttggggtg agtactccct ctcaaaagcg ggcatgactt ctgcgctaag
     3541 attgtcagtt tccaaaaacg aggaggattt gatattcacc tggcccgcgg tgatgccttt
     3601 gagggtggcc gcgtccatct ggtcagaaaa gacaatcttt ttgttgtcaa gcttgaggtg
     3661 tggcaggctt gagatctggc catacacttg agtgacaatg acatccactt tgcctttctc
     3721 tccacaggtg tccactccca ggtccaactg caggtcgagc atgcatctag ggcggccaat
     3781 tccgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     3841 ggtgtgcgtt tgtctatatg tgattttcca ccatattgcc gtcttttggc aatgtgaggg
     3901 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     3961 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     4021 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     4081 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     4141 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     4201 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     4261 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     4321 acggggacgt ggttttcctt tgaaaaacac gatgataagc ttgccacaac ccacaaggag
     4381 acgaccttcc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     4441 ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     4501 cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     4561 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     4621 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     4681 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     4741 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     4801 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     4861 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     4921 cgtcaccgcc gacgtcgagt gcccgaagga ccgcgcgacc tggtgcatga cccgcaagcc
     4981 cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca
     5041 tggctccgac cgaagccgac ccgggcggcc ccgccgaccc cgcacccgcc cccgaggccc
     5101 accgactcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5161 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5221 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5281 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     5341 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgag tgcattctag
     5401 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     5461 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5521 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5581 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5641 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5701 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5761 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5821 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5881 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5941 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6001 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6061 aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6121 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6181 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6241 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6301 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     6361 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6421 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6481 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6541 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6601 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6661 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6721 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6781 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6841 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6901 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6961 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7021 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7081 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7141 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7201 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7261 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7321 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7381 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7441 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7501 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7561 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7621 aaaagtgcca cctgacgtc
//
