LOCUS       sequence-274000-        4235 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal Cleavable Donor Plasmid for tagging with Clover-BlastR 
            using NHEJ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4235)
  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 4235)
  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-Clover-BlastR
FEATURES             Location/Qualifiers
     source          1..4235
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     98..120
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     112..129
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     113..129
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(253..274)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     861..882
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(901..934)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    complement(901..934)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     promoter        942..1441
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(977..1000)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1374..1393
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1460..1858
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(2133..2150)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2139..2363
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2414..3002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2494..2513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3173..3964,3965..4033))
                     /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     3796..3815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4034..4138)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
       61 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      121 acggccagtg aattcgagct cggtacctaa tgatagctag cgggccagta cccaaaaagc
      181 ggggggtctg gtggcagtgg agggggatct gtgagcaagg gcgaggagct gttcaccggg
      241 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtccgc
      301 ggcgagggcg agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc
      361 ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cgtggcctgc
      421 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
      481 ggctacgtcc aggagcgcac catctctttc aaggacgacg gtacctacaa gacccgcgcc
      541 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
      601 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acttcaacag ccactacgtc
      661 tatatcacgg ccgacaagca gaagaactgc atcaaggcta acttcaagat ccgccacaac
      721 gttgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
      781 ggccccgtgc tgctgcccga caaccactac ctgagccatc agtccaagct gagcaaagac
      841 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc ctaaggccgc
      901 ataacttcgt ataggatacc ttatacgaag ttatctcacc cgggtagggg aggcgctttt
      961 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     1021 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     1081 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     1141 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     1201 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     1261 gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
     1321 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg
     1381 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc
     1441 gacagccatc gacggtacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga
     1501 aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc
     1561 agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg
     1621 accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac
     1681 ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg
     1741 acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca
     1801 gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctgagg
     1861 atccctgatg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1921 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1981 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2041 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcacttgtt
     2101 aattgcagct tatagcggcc gccgctgatc agcctcgact gtgccttcta gttgccagcc
     2161 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2221 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2281 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2341 tggggatgcg gtgggctcta tggacgcgtc agcaaaaggc caggaaccgt aaaaaggccg
     2401 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2461 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2521 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2581 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2641 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2701 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2761 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2821 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2881 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2941 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3001 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aacttacgtt
     3061 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3121 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3181 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3241 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3301 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3361 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3421 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3481 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3541 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3601 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3661 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3721 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3781 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3841 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3901 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3961 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4021 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4081 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4141 catttccccg aaaagtgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     4201 tcaggcgcca ttcgccattc aggctgcgca actgt
//
