LOCUS       sequence-267679-        3759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal Cleavable Donor Plasmid for tagging with tdTomato using 
            NHEJ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3759)
  AUTHORS   Artegiani B, Hendriks D, Beumer J, Kok R, Zheng X, Joore I, Chuva de
            Sousa Lopes S, van Zon J, Tans S, Clevers H
  TITLE     Fast and efficient generation of knock-in human organoids using 
            homology-independent CRISPR-Cas9 precision genome editing.
  JOURNAL   Nat Cell Biol. 2020 Mar;22(3):321-331. doi: 
            10.1038/s41556-020-0472-5. Epub 2020 Mar 2.
  PUBMED    32123335
REFERENCE   2  (bases 1 to 3759)
  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"; doi: 
            "10.1038/s41556-020-0472-5"; journalName: "Nat Cell Biol"; date: 
            "2020-03"; volume: "22"; issue: "3"; pages: "321-331"
COMMENT     Sequence Label: pCRISPR-HOT_tdTomato
FEATURES             Location/Qualifiers
     source          1..3759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     415..434
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(653..757)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     952..974
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     966..983
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     967..983
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1065..2492
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYE
                     GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMN
                     FEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVL
                     KGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYER
                     SEGRHHLFLYGMDELYK"
     primer_bind     complement(2511..2528)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2517..2741
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2792..3380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2872..2891)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3551..3759,1..583,584..652))
                     /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"
ORIGIN
        1 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
       61 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
      121 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
      181 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
      241 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
      301 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
      361 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
      421 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
      481 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
      541 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
      601 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
      661 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
      721 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgcggt
      781 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
      841 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
      901 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
      961 gacgttgtaa aacgacggcc agtgaattcg agctcggtac ctaatgatag ctagcgggcc
     1021 agtacccaaa aagcgggggg tctggtggca gtggaggggg atccgtgagc aagggcgagg
     1081 aggtcatcaa agagttcatg cgcttcaagg tgcgcatgga gggctccatg aacggccacg
     1141 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     1201 tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttca
     1261 tgtacggctc caaggcgtac gtgaagcacc ccgccgacat ccccgattac aagaagctgt
     1321 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggtctggtga
     1381 ccgtgaccca ggactcctcc ctgcaggacg gcacgctgat ctacaaggtg aagatgcgcg
     1441 gcaccaactt cccccccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     1501 ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagatccac caggccctga
     1561 agctgaagga cggcggccac tacctggtgg agttcaagac catctacatg gccaagaagc
     1621 ccgtgcaact gcccggctac tactacgtgg acaccaagct ggacatcacc tcccacaacg
     1681 aggactacac catcgtggaa cagtacgagc gctccgaggg ccgccaccac ctgttcctgg
     1741 ggcatggcac cggcagcacc ggcagcggca gctccggcac cgcctcctcc gaggacaaca
     1801 acatggccgt catcaaagag ttcatgcgct tcaaggtgcg catggagggc tccatgaacg
     1861 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     1921 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc
     1981 agttcatgta cggctccaag gcgtacgtga agcaccccgc cgacatcccc gattacaaga
     2041 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggtc
     2101 tggtgaccgt gacccaggac tcctccctgc aggacggcac gctgatctac aaggtgaaga
     2161 tgcgcggcac caacttcccc cccgacggcc ccgtaatgca gaagaagacc atgggctggg
     2221 aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag atccaccagg
     2281 ccctgaagct gaaggacggc ggccactacc tggtggagtt caagaccatc tacatggcca
     2341 agaagcccgt gcaactgccc ggctactact acgtggacac caagctggac atcacctccc
     2401 acaacgagga ctacaccatc gtggaacagt acgagcgctc cgagggccgc caccacctgt
     2461 tcctgtacgg catggacgag ctgtacaagt aagcggccgc cgctgatcag cctcgactgt
     2521 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2581 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2641 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2701 agacaatagc aggcatgctg gggatgcggt gggctctatg gacgcgtcag caaaaggcca
     2761 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2821 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2881 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2941 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3001 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3061 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3121 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3181 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     3241 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3301 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3361 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3421 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3481 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3541 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3601 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3661 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3721 cagcaataaa ccagccagcc ggaagggccg agcgcagaa
//
