LOCUS       sequence-8216-ex        7677 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 7677)
  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 7677)
  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: M76 pIRESpuro-GLUE-hDsh2
FEATURES             Location/Qualifiers
     source          1..7677
                     /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"
     intron          3537..3765
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     3748..3767
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(3823..4397,4398..4400,4401..4409)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3990..4007)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4217..4236
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4429..5028
                     /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(4429..4448)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4925..4945
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5164..5181)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5170..5394
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5441..5460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5520..5536)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5520..5536)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5533..5555)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5544..5560
                     /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(5568..5574,5575..5592,5593..5598))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5613..5634
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5751..5768)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5922..6510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6002..6021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6681..7472,7473..7541))
                     /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     7304..7323
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7542..7646)
                     /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 gcgcgccaat ggcgggtagc agcactgggg gcggtggggt
     1261 tggggagacg aaggtgattt accacctgga tgaggaagag actccctacc tggtgaagat
     1321 ccctgtcccc gccgagcgca tcaccctcgg cgatttcaag agcgtcctgc agcggcccgc
     1381 gggcgccaag tactttttca agtctatgga tcaggatttc ggggtggtga aggaagaaat
     1441 ttcagatgac aacgcccgcc tcccctgctt caacggaagg gtggtatcct ggctggtgtc
     1501 ctcagataat ccccaacccg agatggcccc tccagtccat gagcctcggg cagaactggc
     1561 gcctccagcc ccacctttac ctcctttgcc acccgagagg accagcggca ttggggactc
     1621 aaggcctcca tccttccacc ctaatgtgtc cagcagccat gagaatctgg agcctgagac
     1681 agaaaccgag tcagtagtgt cactgaggcg ggagcggcct cgcaggagag acagcagtga
     1741 gcatggcgct gggggccaca ggactggtgg cccctcaagg ctggagcgcc acctggccgg
     1801 atacgagagc tcctctaccc tcatgaccag cgagctggag agtaccagcc tgggggactc
     1861 ggacgaggag gacaccatga gcaggttcag cagctccacg gagcagagca gtgcctcccg
     1921 cctccttaag cgccaccggc ggcgaaggaa gcagaggcca ccccgcctgg agaggacgtc
     1981 atccttcagc agcgtcacag attccacaat gtctctcaat atcatcacag tcacgctaaa
     2041 catggagaag tacaacttcc tgggtatctc cattgttggc cagagcaatg agcggggaga
     2101 cggaggcatc tacattggct ccatcatgaa gggtggggct gtggcggccg acgggcgcat
     2161 tgagccaggg gacatgcttt tgcaggtgaa tgacatgaac tttgagaaca tgagcaacga
     2221 tgacgctgtg cgggtgctga gggacattgt gcacaagcct ggccccattg tgctgactgt
     2281 ggccaagtgc tgggatccct ctcctcaggc ctatttcact ctcccccgaa atgagcccat
     2341 ccagccaatt gaccctgctg cctgggtgtc ccattccgcg gctctgactg gcaccttccc
     2401 agcctatcca ggttcctcct ccatgagcac cattacatct ggatcgtctt tgcctgatgg
     2461 ctgtgaaggc cggggtctct ccgtccatac ggacatggca tcggtgacca aggccatggc
     2521 agctccagag tctggactgg aagtccggga ccgcatgtgg ctcaagatca ccatccctaa
     2581 tgcctttctg ggctcggatg tggttgactg gctctaccat cacgtggagg gctttcctga
     2641 gcggcgggag gcccgcaagt atgccagcgg gctgctcaaa gcaggcctga tccgacacac
     2701 cgtcaacaag atcaccttct ctgagcagtg ctattacgtc ttcggagacc tcagtggtgg
     2761 ctgtgagagc tacctagtca acctgtctct caatgacaac gatggctcca gtggggcttc
     2821 agaccaggat accctggctc ctctgcctgg ggccaccccc tggcccctgc tgcccacttt
     2881 ctcctaccaa taccctgccc cacaccccta cagcccgcag cctccaccct accatgagct
     2941 ttcatcttac acctatggtg ggggcagtgc cagcagccag catagtgagg gcagccggag
     3001 cagtgggtcg acacggagtg atgggggggc agggcgcacg gggaggcccg aggagcgggc
     3061 ccccgagtcc aagtccggca gtggcagtga gtctgagccc tccagccgag ggggcagcct
     3121 tcggcggggt ggggaagcaa gtgggactag cgatgggggc cctcctccat ccagaggctc
     3181 aactgggggt gcccctaatc tccgagccca cccagggctc catccctatg gaccgccccc
     3241 tggcatggcc ctcccctaca accccatgat ggtggtcatg atgcccccac ctccacctcc
     3301 agtccctcca gcagtgcagc ctccgggggc ccctccagtc agagacctgg gctctgtgcc
     3361 cccagaactg acagccagcc gccaaagctt ccacatggcc atgggcaatc ccagcgagtt
     3421 ctttgtggat gttatgtagg cggccgcgtc gacggaattc gcccttttcg gatccactag
     3481 taacggccgc cagtgtgctg gaattaattc gctgtctgcg agggccagct gttggggtga
     3541 gtactccctc tcaaaagcgg gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga
     3601 ggaggatttg atattcacct ggcccgcggt gatgcctttg agggtggccg cgtccatctg
     3661 gtcagaaaga caatcttttt gttgtcaagc ttgaggtgtg gcaggcttga gatctggcca
     3721 tacacttgag tgacaatgac atccactttg cctttctctc cacaggtgtc cactcccagg
     3781 tccaactgca ggtcgagcat gcatctaggg cggccaattc cgcccctctc cctccccccc
     3841 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtg
     3901 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     3961 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     4021 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
     4081 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     4141 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     4201 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     4261 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt
     4321 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     4381 aaaaacacga tgataagctt gccacaaccc acaaggagac gaccttccat gaccgagtac
     4441 aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg caccctcgcc
     4501 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg ccacatcgag
     4561 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     4621 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     4681 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     4741 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     4801 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     4861 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     4921 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgagtgc
     4981 ccgaaggacc gcgcgacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac
     5041 gacccgcagc gcccgaccga aaggagcgca cgaccccatg gctccgaccg aagccgaccc
     5101 gggcggcccc gccgaccccg cacccgcccc cgaggcccac cgactctaga gctcgctgat
     5161 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     5221 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     5281 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     5341 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     5401 aggcggaaag aaccagctgg ggctcgagtg cattctagtt gtggtttgtc caaactcatc
     5461 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5521 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5581 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5641 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5701 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5761 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5821 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5881 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5941 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6001 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6061 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc
     6121 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6181 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6241 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6301 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6361 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6421 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6481 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6541 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6601 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6661 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6721 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6781 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6841 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6901 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6961 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7021 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7081 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7141 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7201 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7261 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7321 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7381 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7441 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7501 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7561 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7621 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
