LOCUS       sequence-305646-        5257 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology Directed Repair construct for N-terminal tagging of 
            hsCTNNB1 with mScarlet-i.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5257)
  AUTHORS   de Man SM, Zwanenburg G, van der Wal T, Hink MA, van Amerongen R
  TITLE     Quantitative live-cell imaging and computational modeling shed new 
            light on endogenous WNT/CTNNB1 signaling dynamics.
  JOURNAL   Elife. 2021 Jun 30;10. pii: 66440. doi: 10.7554/eLife.66440.
  PUBMED    34190040
REFERENCE   2  (bases 1 to 5257)
  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: "Elife."; 
            date: "2021-06-30"; volume: "10. pii: 66440. doi"; pages: " 
            10.7554/eLife.66440"
COMMENT     Sequence Label: pRepair-mScI-CTNNB1
FEATURES             Location/Qualifiers
     source          1..5257
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..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(join(4145..4151,4152..4169,4170..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
//
