LOCUS Exported 7639 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS M77 pIRESpuro-GLUE-hDsh3 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7639 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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 /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 3785..4358 /label=IRES /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(5530..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(6643..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 //