LOCUS Exported 5257 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Homology Directed Repair construct for N-terminal tagging of hsCTNNB1 with mScarlet-i. ACCESSION . VERSION . KEYWORDS pRepair-mScI-CTNNB1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5257) AUTHORS de Man SMA, Zwanenburg G, Hink MA, van Amerongen R TITLE Quantitative live-cell imaging yields novel insight into endogenous WNT/CTNNB1 signaling dynamics JOURNAL bioRxiv REFERENCE 2 (bases 1 to 5257) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5257 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(1..861) /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 624..643 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(862..966) /gene="bla" /label=AmpR promoter rep_origin complement(992..1447) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1129..1150) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1341..1360 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1574..1596 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 1588..1605 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 1589..1605 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 1612..1631 /label=T7 /note="T7 promoter, forward primer" promoter 1612..1630 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 2450..3145 /codon_start=1 /product="bright monomeric red fluorescent protein evolved from a synthetic template (Bindels et al., 2016)" /label=mScarlet-I /note="matures faster than mScarlet" /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH STGGMDELYK" primer_bind complement(2583..2601) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2851..2870 /label=mCherry-F /note="mCherry, forward primer" CDS 3635..3670 /codon_start=1 /product="nanobody epitope tag corresponding to amino acids 16-27 of human beta-catenin (Traenkle et al., 2015)" /label=BC2 tag /translation="PDRKAAVSHWQQ" misc_feature 3939..4046 /label=MCS /note="pBluescript multiple cloning site" primer_bind 3963..3979 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 3963..3979 /label=pBluescriptSK /note="For pBluescript vector" primer_bind complement(4013..4029) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4014..4030) /label=pBluescriptKS /note="For pBluescript vector" promoter complement(4058..4076) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4097..4113) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4097..4113) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4110..4132) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4121..4137 /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(4145..4175) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4190..4211 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4328..4345) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4499..5087) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4579..4598) /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 961 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa 1021 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa 1081 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac 1141 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag 1201 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt 1261 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg 1321 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca 1381 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag 1441 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc 1501 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta 1561 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac 1621 tcactatagg gcgaattgga gctgatgcag tttttttcaa tattgcacaa taaaggcaaa 1681 atacggacaa attagatgat aagatttata taaattttta aaatattgat caaaatatgt 1741 atccatattg gtaatatttg tatttataat aaatcattgc tgtaaatttg aacttagaaa 1801 aattttacta ataaaggtgc ttttgtgttg caaactttca tttgaaaagt aatttttctt 1861 tgtaccaaaa aatctaaaat tcgctattct agtcaccaaa atttgcttta tgaaaaataa 1921 tttttgatgg cactatatca gaaaacaact tgttaaagaa aatgtggagt ttttaaaatc 1981 ccactgtacc tctgttatcc aaaggggatc tgtgaatttt tctgtgaaag gttaaaaaag 2041 gagagacctt taggaattca gagagcagct gatttttgaa tagtgttttc ccctccctgg 2101 cttttattat tacaactctg tgctttttca tcaccatcct gaatatctat aattaatatt 2161 tatactatta ataaaaagac atttttggta aggaggagtt ttcactgaag ttcagcagtg 2221 atggagctgt ggttgaggtg tctggaggag accatgaggt ctgcgtttca ctaacctggt 2281 aaaagaggat atgggttttt tttgtgggtg taatagtgac atttaacagg tatcccagtg 2341 acttaggagt attaatcaag ctaaatttaa atcctaatga cttttgatta acttttttta 2401 gggtatttga agtataccat acaactgttt tgaaaatcca gcgtggacaa tggtgagcaa 2461 gggcgaggca gtgatcaagg agttcatgcg gttcaaggtg cacatggagg gctccatgaa 2521 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac 2581 cgccaagctg aaggtgacca agggtggccc cctgcccttc tcctgggaca tcctgtcccc 2641 tcagttcatg tacggctcca gggccttcat caagcacccc gccgacatcc ccgactacta 2701 taagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg 2761 cgccgtgacc gtgacccagg acacctccct ggaggacggc accctgatct acaaggtgaa 2821 gctccgcggc accaacttcc ctcctgacgg ccccgtaatg cagaagaaga caatgggctg 2881 ggaagcgtcc accgagcggt tgtaccccga ggacggcgtg ctgaagggcg acattaagat 2941 ggccctgcgc ctgaaggacg gcggccgcta cctggcggac ttcaagacca cctacaaggc 3001 caagaagccc gtgcagatgc ccggcgccta caacgtcgac cgcaagttgg acatcacctc 3061 ccacaacgag gactacaccg tggtggaaca gtacgaacgc tccgagggcc gccactccac 3121 cggcggcatg gacgagctgt acaagatggc tactcaaggt ttgtgtcatt aaatctttag 3181 ttactgaatt ggggctctgc ttcgttgcca ttaagccagt ctggctgaga tccccctgct 3241 ttcctctctc cctgcttact tgtcaggcta ccttttgctc cattttctgc tcactcctcc 3301 taatggcttg gtgaaatagc aaacaagcca ccagcaggaa tctagtctgg atgactgctt 3361 ctggagcctg gatgcagtac cattcttcca ctgattcagt gagtaactgt taggtggttc 3421 cctaagggat taggtatttc atcactgagc taaccctggc tatcattctg cttttcttgg 3481 ctgtctttca gatttgactt tatttctaaa aatatttcaa tgggtcatat cacagattct 3541 ttttttttaa attaaagtaa catttccaat ctactaatgc taatactgtt tcgtatttat 3601 agctgatttg atggagttgg acatggccat ggaaccagac agaaaagcgg ctgttagtca 3661 ctggcagcaa cagtcttacc tggactctgg aatccattct ggtgccacta ccacagctcc 3721 ttctctgagt ggtaaaggca atcctgagga agaggatgtg gatacctccc aagtcctgta 3781 tgagtgggaa cagggatttt ctcagtcctt cactcaagaa caagtagctg gtaagagtat 3841 tatttttcat tgccttactg aaagtcagaa tgcagttttg agaactaaaa agttagtgta 3901 taatagttta aataaaatgt tgtggtgaag aaaagagaga gctccaccgc ggtggcggcc 3961 gctctagaac tagtggatcc cccgggctgc aggaattcga tatcaagctt atcgataccg 4021 tcgacctcga gggggggccc ggtaccagct tttgttccct ttagtgaggg ttaatttcga 4081 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 4141 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 4201 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 4261 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 4321 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4381 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4441 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4501 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4561 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 4621 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 4681 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 4741 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 4801 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 4861 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 4921 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 4981 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 5041 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5101 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5161 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 5221 caatctaaag tatatatgag taaacttggt ctgacag //