LOCUS       sequence-273998-        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     77..99
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     91..108
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     92..108
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             190..1617
                     /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(1636..1653)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1642..1866
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(1917..2505)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1997..2016)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2676..3467,3468..3536))
                     /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     3299..3318
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3537..3641)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
       61 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgagctc
      121 ggtacctaat gatagctagc gggccagtac ccaaaaagcg gggggtctgg tggcagtgga
      181 gggggatccg tgagcaaggg cgaggaggtc atcaaagagt tcatgcgctt caaggtgcgc
      241 atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
      301 tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc
      361 tgggacatcc tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc
      421 gacatccccg attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
      481 aacttcgagg acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg
      541 ctgatctaca aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag
      601 aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg
      661 aagggcgaga tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc
      721 aagaccatct acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc
      781 aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc
      841 gagggccgcc accacctgtt cctggggcat ggcaccggca gcaccggcag cggcagctcc
      901 ggcaccgcct cctccgagga caacaacatg gccgtcatca aagagttcat gcgcttcaag
      961 gtgcgcatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1021 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc
     1081 ttcgcctggg acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac
     1141 cccgccgaca tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc
     1201 gtgatgaact tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac
     1261 ggcacgctga tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta
     1321 atgcagaaga agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc
     1381 gtgctgaagg gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg
     1441 gagttcaaga ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg
     1501 gacaccaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag
     1561 cgctccgagg gccgccacca cctgttcctg tacggcatgg acgagctgta caagtaagcg
     1621 gccgccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     1681 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     1741 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     1801 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     1861 ctatggacgc gtcagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1921 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1981 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2041 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2101 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2161 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2221 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2281 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2341 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2401 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2461 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2521 ttttctacgg ggtctgacgc tcagtggaac gaaaacttac gttaagggat tttggtcatg
     2581 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2641 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2701 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2761 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     2821 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     2881 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     2941 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3001 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3061 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3121 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3181 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3241 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3301 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3361 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3421 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3481 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3541 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3601 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3661 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca
     3721 ttcaggctgc gcaactgttg ggaagggcga tcggtgcgg
//
