LOCUS       sequence-305620-        5278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology Directed Repair construct for N-terminal tagging of 
            hsCTNNB1 with SGFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5278)
  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 5278)
  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-SGFP2-CTNNB1
FEATURES             Location/Qualifiers
     source          1..5278
                     /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             join(2450..2452,2453..2455,2456..3166)
                     /codon_start=1
                     /product="strongly enhanced green fluorescent protein 
                     (Kremers et al., 2007)"
                     /label=SGFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2495..2516)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2756..2775)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3103..3124
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3656..3691
                     /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    3960..4067
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     3984..4000
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3984..4000
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4034..4050)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4035..4051)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(4079..4097)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4118..4134)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4118..4134)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4131..4153)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4142..4158
                     /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(4166..4172,4173..4190,4191..4196))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4211..4232
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4349..4366)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4520..5108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4600..4619)
                     /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 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     2521 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     2581 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     2641 cctgacctac ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     2701 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2761 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2821 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2881 caactacaac agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc
     2941 caacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca
     3001 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     3061 ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3121 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagatgg ctactcaagg
     3181 tttgtgtcat taaatcttta gttactgaat tggggctctg cttcgttgcc attaagccag
     3241 tctggctgag atccccctgc tttcctctct ccctgcttac ttgtcaggct accttttgct
     3301 ccattttctg ctcactcctc ctaatggctt ggtgaaatag caaacaagcc accagcagga
     3361 atctagtctg gatgactgct tctggagcct ggatgcagta ccattcttcc actgattcag
     3421 tgagtaactg ttaggtggtt ccctaaggga ttaggtattt catcactgag ctaaccctgg
     3481 ctatcattct gcttttcttg gctgtctttc agatttgact ttatttctaa aaatatttca
     3541 atgggtcata tcacagattc ttttttttta aattaaagta acatttccaa tctactaatg
     3601 ctaatactgt ttcgtattta tagctgattt gatggagttg gacatggcca tggaaccaga
     3661 cagaaaagcg gctgttagtc actggcagca acagtcttac ctggactctg gaatccattc
     3721 tggtgccact accacagctc cttctctgag tggtaaaggc aatcctgagg aagaggatgt
     3781 ggatacctcc caagtcctgt atgagtggga acagggattt tctcagtcct tcactcaaga
     3841 acaagtagct ggtaagagta ttatttttca ttgccttact gaaagtcaga atgcagtttt
     3901 gagaactaaa aagttagtgt ataatagttt aaataaaatg ttgtggtgaa gaaaagagag
     3961 agctccaccg cggtggcggc cgctctagaa ctagtggatc ccccgggctg caggaattcg
     4021 atatcaagct tatcgatacc gtcgacctcg agggggggcc cggtaccagc ttttgttccc
     4081 tttagtgagg gttaatttcg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4141 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4201 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4261 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4321 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4381 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4441 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4501 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4561 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4621 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4681 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4741 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4801 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4861 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4921 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4981 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5041 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5101 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5161 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5221 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacag
//
