LOCUS       sequence-267680-        4279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal Cleavable Donor Plasmid for tagging with P2A-Clover-BlastR
            using NHEJ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4279)
  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 4279)
  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-P2A-Clover-BlastR
FEATURES             Location/Qualifiers
     source          1..4279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     427..446
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(665..769)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     964..986
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     978..995
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     979..995
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1086..1142
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(1185..1206)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1793..1814
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1833..1866)
                     /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(1833..1866)
                     /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        1874..2373
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1909..1932)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2306..2325
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2392..2790
                     /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(3043..3060)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3049..3273
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3324..3912)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3404..3423)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4083..4279,1..595,596..664))
                     /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 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
       61 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
      121 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
      181 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
      241 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
      301 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
      361 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
      421 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
      481 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
      541 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
      601 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
      661 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
      721 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
      781 gaaaagtgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      841 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      901 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      961 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctaatgat
     1021 agctagcggg ccagtaccca aaaagcgggg ggtctggtgg cagtggaggg ggatccggaa
     1081 gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     1141 ctgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1201 gcgacgtaaa cggccacaag ttcagcgtcc gcggcgaggg cgagggcgat gccaccaacg
     1261 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1321 tcgtgaccac cttcggctac ggcgtggcct gcttcagccg ctaccccgac cacatgaagc
     1381 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatctctt
     1441 tcaaggacga cggtacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1501 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1561 agctggagta caacttcaac agccactacg tctatatcac ggccgacaag cagaagaact
     1621 gcatcaaggc taacttcaag atccgccaca acgttgagga cggcagcgtg cagctcgccg
     1681 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1741 acctgagcca tcagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1801 tgctggagtt cgtgaccgcc gcctaaggcc gcataacttc gtataggata ccttatacga
     1861 agttatctca cccgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     1921 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     1981 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
     2041 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
     2101 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
     2161 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
     2221 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     2281 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     2341 ctgttctcct cttcctcatc tccgggcctt tcgacagcca tcgacggtac catggccaag
     2401 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
     2461 cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
     2521 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
     2581 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
     2641 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
     2701 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
     2761 ctgccctctg gttatgtgtg ggagggctga ggatccctga tgtgccttct agttgccagc
     2821 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     2881 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     2941 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3001 ctggggatgc ggtgggctct atgggcggcc gccgctgatc agcctcgact gtgccttcta
     3061 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3121 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3181 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3241 gcaggcatgc tggggatgcg gtgggctcta tggacgcgtc agcaaaaggc caggaaccgt
     3301 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3361 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3421 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3481 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3541 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3601 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3661 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3721 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     3781 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3841 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3901 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3961 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4021 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4081 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4141 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4201 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4261 aaccagccag ccggaaggg
//
