LOCUS       sequence-331132-        5934 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tp63 reporter targeting repair template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5934)
  AUTHORS   Sun D, Evans L, Perrone F, Sokleva V, Lim K, Rezakhani S, Lutolf M, 
            Zilbauer M, Rawlins EL
  TITLE     A functional genetic toolbox for human tissue-derived organoids.
  JOURNAL   eLife. 2021 Oct 6;10. pii: 67886. doi: 10.7554/eLife.67886.
  PUBMED    34612202
REFERENCE   2  (bases 1 to 5934)
  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: "2021-10-6"; volume: "10. pii: 67886. doi"; pages: " 
            10.7554/eLife.67886"
COMMENT     Sequence Label: TP63-T2A-Rox-EF1a-Rox-Venus-NLS repair template
FEATURES             Location/Qualifiers
     source          1..5934
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(1928..1948)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(1949..2659,2660..2662))
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1991..2012)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2340..2359
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2599..2620
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2669..2700
                     /label=rox
                     /bound_moiety="Dre recombinase"
     LTR             2732..3000
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     promoter        complement(3013..3224)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    3257..3288
                     /label=rox
                     /bound_moiety="Dre recombinase"
     CDS             complement(3296..3349)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(4309..4326)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4309..4325)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4318..4340)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4534..4553)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4653..4675
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4713..4731)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4799..4903
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4904..4972,4973..5764)
                     /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     complement(5122..5141)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccaatattgt aagataaggt atctggtatt
     1021 aaatttaagg gggttactga tatgctcaaa tacatgacgt cgggtgtttt tgaactggct
     1081 gctatgggcc ttttaagaat tttcctcatt cccaaccaaa aactaaagag gacgtttgga
     1141 catgaacttc ttcttataac tggattaaat caccccaacg tgaaccatca tcactagaag
     1201 aaatgtcagt aatataaaat ataccaacac tgagaaaaga atacatatct acagtcttac
     1261 ctacataata agcaatggaa atggatctac tcaaaggttt gatgtggcat gtatttagaa
     1321 tccaaagaac tgaaagaatc tgtacatcat cgtcactcac atgtagcatt aagctctaga
     1381 tacctatcac catacatgat accccttaag ggaaggagta tagacaagcc atccaaaaca
     1441 agtaaattaa aaaatggtca ataagtgcaa ctcaattttc tttcatttaa attacatttt
     1501 aaacaatact caatgccccc cacccaaaaa tctatactgt atatttatac atttatatat
     1561 atagtcttag actataaggg tgaacatgct ttataaaagt caatgagaaa tctctgcagc
     1621 ttaaggagac acccaactct tttttgctca cttctaccta gccatggcag ctacagttct
     1681 gcaagctata gttttgaagg cttaaagcca gaatcagaat tagatgccag gtcagatgtt
     1741 aggtaagagg tagcctctta cttctccttc ccctaagaaa tcagacaaga ggaaggggag
     1801 gagctaggga gaaggctttg aagattaagc aggagtgctt ttagggggct ggcagttagg
     1861 agagggatag gaagagctca catggtgagg ctcactcccc ctcctctttg atgcgctgtt
     1921 gcttttatac ctttctcttc ttttttggct tgtacagctc gtccatgccg agagtgatcc
     1981 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc
     2041 tcagggcgga ctggtagctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga
     2101 tgggggtgtt ctgctggtag tggtcggcga gctgcacgcc gccgtcctcg atgttgtggc
     2161 ggatcttgaa gttggccttg atgccgttct tctgcttgtc ggcggtgata tagacgttgt
     2221 ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga
     2281 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg
     2341 tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca
     2401 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcgggcg aagcactgca
     2461 ggccgtagcc cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc
     2521 agatcagctt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc
     2581 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga
     2641 acagctcctc gcccttgctc acggatccta actttaaata attggcatta tttaaagtta
     2701 tcatggtggc gtaggcgccg gtcacagctt ggatctgtaa cggcgcagaa cagaaaacga
     2761 aacaaagacg tagagttgag caagcagggt caggcaaagc gtggagagcc ggctgagtct
     2821 aggtaggctc caagggagcg ccggacaaag gcccggtctc gacctgagct ttaaacttac
     2881 ctagacggcg gacgcagttc aggaggcacc acaggcggga ggcggcagaa cgcgactcaa
     2941 ccggcgtgga tggcggcctc aggtagggcg gcgggcgcgt gaaggagaga tgcgagcccc
     3001 tcgaagcttc agctgtgttc tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac
     3061 tactgcactt atatacggtt ctcccccacc ctcgggaaaa aggcggagcc agtacacgac
     3121 atcactttcc cagtttaccc cgcgccacct tctctaggca cccgttcaat tgccgacccc
     3181 tccccccaac ttctcgggga ctgtgggcga tgtgcgctct gcccactgac gggcaccgga
     3241 gcgatcgcag atcctttaac tttaaataat tggcattatt taaagttata ccggtagggc
     3301 cgggattctc ctccacgtca ccgcatgtta gaagacttcc tctgccctct ccggatcctt
     3361 cgccctcttc cttaattctc tgctgtttat tgcgtcgagc atccatgtca aagttgaagt
     3421 cattccactc atctcggggt gggaaagaga tggtctggcg gagggtgaat cgcacagcat
     3481 caataacacg ctcaccccgg gtctcactgg agcccacact gactgtagag gcactgcttg
     3541 gggtccgcag gagatgagaa ggggaggaga attcgtggag ctgccggtgg tccaggatgc
     3601 ccttccagat cgcatgtcga aattgctcag ggattttcag acttgccaga tcctgtgcaa
     3661 caggggaagg tgtcatggag aatgaaaaca gggagaaaaa cggacattta gtccacagca
     3721 tcctatcatt ccctgatgct ctggtttaat tgatctgaat ctattgatcc acaaattaga
     3781 aaataaaatt agttctctga gtcttcaact ctatggaact aagcacaaag gcacagcctt
     3841 gaggtgtctt tcctgcctgt gtagaacact tcacttagtc tctactggcc tcagtttctt
     3901 cattggtaaa attgggataa tagctagatg agagggcaaa aatcccaaat agaaacatga
     3961 ggtacctact tatgcttgaa tttacctggc tagtcactag aagaaaggta ttaatggaat
     4021 gtcaccacca cttggttgag gagatgagaa gaaaagattg agtactttct atgtgccagg
     4081 ccctgaatta ttttatattc atgatatcat ttaatcccca gattagtggt aaacattatt
     4141 agacctgttt tatagataga acaactgaag gccagagagg ttgacaagtt taaagtcaca
     4201 taactaagtg aaaaaattag ggacttaggg catcctcaga acctgaaacg gcacttttat
     4261 ctaaaaggtc tcttggtgtt tgaataaagc aaggtatcaa aatgcagaac tggccgtcgt
     4321 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     4381 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     4441 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     4501 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4561 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4621 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4681 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     4741 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     4801 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4861 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4921 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4981 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5041 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5101 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5161 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5221 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5281 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5341 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5401 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5461 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5521 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5581 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5641 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5701 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5761 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5821 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5881 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
