LOCUS       sequence-297706-        5282 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 5282)
  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 5282)
  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..5282
                     /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     1615..1634
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1615..1633
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2453..2455,2456..2458,2459..3169)
                     /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(2498..2519)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2759..2778)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3106..3127
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3659..3694
                     /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    3963..4070
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     3987..4003
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3987..4003
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4037..4053)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4038..4054)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(4083..4103)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4083..4101)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4122..4138)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4122..4138)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4135..4157)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4146..4162
                     /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(4170..4176,4177..4194,4195..4200))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4215..4236
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4353..4370)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4524..5112)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4604..4623)
                     /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 ccagtgagcg cgcgtaatac
     1621 gactcactat agggcgaatt ggagctgatg cagttttttt caatattgca caataaaggc
     1681 aaaatacgga caaattagat gataagattt atataaattt ttaaaatatt gatcaaaata
     1741 tgtatccata ttggtaatat ttgtatttat aataaatcat tgctgtaaat ttgaacttag
     1801 aaaaatttta ctaataaagg tgcttttgtg ttgcaaactt tcatttgaaa agtaattttt
     1861 ctttgtacca aaaaatctaa aattcgctat tctagtcacc aaaatttgct ttatgaaaaa
     1921 taatttttga tggcactata tcagaaaaca acttgttaaa gaaaatgtgg agtttttaaa
     1981 atcccactgt acctctgtta tccaaagggg atctgtgaat ttttctgtga aaggttaaaa
     2041 aaggagagac ctttaggaat tcagagagca gctgattttt gaatagtgtt ttcccctccc
     2101 tggcttttat tattacaact ctgtgctttt tcatcaccat cctgaatatc tataattaat
     2161 atttatacta ttaataaaaa gacatttttg gtaaggagga gttttcactg aagttcagca
     2221 gtgatggagc tgtggttgag gtgtctggag gagaccatga ggtctgcgtt tcactaacct
     2281 ggtaaaagag gatatgggtt ttttttgtgg gtgtaatagt gacatttaac aggtatccca
     2341 gtgacttagg agtattaatc aagctaaatt taaatcctaa tgacttttga ttaacttttt
     2401 ttagggtatt tgaagtatac catacaactg ttttgaaaat ccagcgtgga caatggtgag
     2461 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     2521 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2581 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2641 caccctgacc tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga
     2701 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2761 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2821 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2881 gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga acggcatcaa
     2941 ggccaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg ccgaccacta
     3001 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     3061 cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3121 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaaga tggctactca
     3181 aggtttgtgt cattaaatct ttagttactg aattggggct ctgcttcgtt gccattaagc
     3241 cagtctggct gagatccccc tgctttcctc tctccctgct tacttgtcag gctacctttt
     3301 gctccatttt ctgctcactc ctcctaatgg cttggtgaaa tagcaaacaa gccaccagca
     3361 ggaatctagt ctggatgact gcttctggag cctggatgca gtaccattct tccactgatt
     3421 cagtgagtaa ctgttaggtg gttccctaag ggattaggta tttcatcact gagctaaccc
     3481 tggctatcat tctgcttttc ttggctgtct ttcagatttg actttatttc taaaaatatt
     3541 tcaatgggtc atatcacaga ttcttttttt ttaaattaaa gtaacatttc caatctacta
     3601 atgctaatac tgtttcgtat ttatagctga tttgatggag ttggacatgg ccatggaacc
     3661 agacagaaaa gcggctgtta gtcactggca gcaacagtct tacctggact ctggaatcca
     3721 ttctggtgcc actaccacag ctccttctct gagtggtaaa ggcaatcctg aggaagagga
     3781 tgtggatacc tcccaagtcc tgtatgagtg ggaacaggga ttttctcagt ccttcactca
     3841 agaacaagta gctggtaaga gtattatttt tcattgcctt actgaaagtc agaatgcagt
     3901 tttgagaact aaaaagttag tgtataatag tttaaataaa atgttgtggt gaagaaaaga
     3961 gagagctcca ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat
     4021 tcgatatcaa gcttatcgat accgtcgacc tcgagggggg gcccggtacc cagcttttgt
     4081 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     4141 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4201 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4261 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4321 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4381 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4441 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4501 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4561 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4621 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4681 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4741 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4801 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4861 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4921 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     4981 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5041 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5101 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5161 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5221 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5281 ag
//
