LOCUS       sequence-481917-        3909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR-HOT donor plasmid for mNeonGreen fusion protein co-expressing
            membrane-bound mCherry via a P2A-T2A tandem.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3909)
  AUTHORS   Krueger D, Spoelstra WK, Mastebroek DJ, Kok RNU, Wu S, Nikolaev M, 
            Bannier-Helaouet M, Gjorevski N, Lutolf M, van Es J, van Zon J, Tans
            SJ, Clevers H
  TITLE     Epithelial tension controls intestinal cell extrusion.
  JOURNAL   Science. 2025 Sep 4;389(6764):eadr8753. doi: 
            10.1126/science.adr8753. Epub 2025 Sep 4.
  PUBMED    40906841
REFERENCE   2  (bases 1 to 3909)
  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: "Science."; 
            date: "2025-09-4"; volume: "389(6764):eadr8753. doi"; pages: " 
            10.1126/science.adr8753. Epub 2025 Sep 4"
COMMENT     Sequence Label: pCRISPR-HOT-mNeon-P2A-T2A-mCherry-CAAX
FEATURES             Location/Qualifiers
     source          1..3909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..70
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     62..79
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     63..79
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             161..862
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEEL
                     NLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
                     SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTF
                     KWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKA
                     FTDVMGMDELYK"
     regulatory      410..419
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             863..916
                     /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"
     CDS             926..982
                     /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"
     CDS             983..1687
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1125..1143)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1393..1412
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1757..1774)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1763..1987
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2038..2626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2118..2137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2797..3588,3589..3657))
                     /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     3420..3439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3658..3762)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
       61 ttgtaaaacg acggccagtg aattcgagct cggtacctaa tgatagctag cgggccagta
      121 cccaaaaagc ggggggtctg gtggcagtgg agggggatcc gtgagcaagg gcgaggagga
      181 taacgcctct ctcccagcga cacatgagtt acacatcttt ggctccatca acggtgtgga
      241 ctttgacatg gtgggtcagg gcaccggcaa tccaaatgat ggttatgagg agttaaacct
      301 gaagtccacc aagggtgacc tccagttctc cccctggatt ctggtccctc atatcgggta
      361 tggcttccat cagtacctgc cctaccctga cgggatgtcg cctttccagg ccgccatggt
      421 agatggctcc ggataccaag tccatcgcac aatgcagttt gaagatggtg cctcccttac
      481 tgttaactac cgctacacct acgagggaag ccacatcaaa ggagaggccc aggtgaaggg
      541 gactggtttc cctgctgacg gtcctgtgat gaccaactcg ctgaccgctg cggactggtg
      601 caggtcgaag aagacttacc ccaacgacaa aaccatcatc agtaccttta agtggagtta
      661 caccactgga aatggcaagc gctaccggag cactgcgcgg accacctaca cctttgccaa
      721 gccaatggcg gctaactatc tgaagaacca gccgatgtac gtgttccgta agacggagct
      781 caagcactcc aagaccgagc tcaacttcaa ggagtggcaa aaggccttta ccgatgtgat
      841 gggcatggac gagctgtaca aggagggcag aggaagtctg ctaacatgcg gtgacgtcga
      901 ggagaatcct ggcccaggat ccggagctac taacttcagc ctgctgaagc aggctggaga
      961 cgtggaggag aaccctggac ctgtgagcaa gggcgaggag gataacatgg ccatcatcaa
     1021 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
     1081 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     1141 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc
     1201 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
     1261 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     1321 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
     1381 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg
     1441 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga
     1501 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct
     1561 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac
     1621 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct
     1681 gtacaagggt aaaaagaaga aaaagaagtc aaagacaaag tgtgtaatta tgtagtaggc
     1741 ggccgccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     1801 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     1861 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     1921 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     1981 tctatggacg cgtcagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2041 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2101 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2161 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2221 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2281 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2341 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2401 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2461 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2521 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2581 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2641 cttttctacg gggtctgacg ctcagtggaa cgaaaactta cgttaaggga ttttggtcat
     2701 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     2761 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     2821 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     2881 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     2941 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3001 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3061 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3121 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     3181 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     3241 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     3301 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     3361 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     3421 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     3481 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     3541 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     3601 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     3661 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3721 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     3781 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc
     3841 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     3901 gctggcgaa
//
