LOCUS       sequence-481729-        4613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR-HOT donor plasmid for mNeonGreen fusion protein co-expressing
            membrane-bound mCherry via PGK promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4613)
  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 4613)
  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-Stop-PGK-mCherry-CAAX
FEATURES             Location/Qualifiers
     source          1..4613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(19..607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(99..118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(778..1569,1570..1638))
                     /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     1401..1420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1639..1743)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1938..1960
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1952..1969
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1953..1969
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2051..2755
                     /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      2300..2309
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2780..3004
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3037..3536
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3072..3095)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3469..3488
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      3568..3577
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3574..4281
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3719..3737)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3987..4006
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4357..4581
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
       61 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      121 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      181 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
      241 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
      301 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      361 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
      421 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
      481 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
      541 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
      601 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactt
      661 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
      721 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
      781 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
      841 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      901 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
      961 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     1021 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     1081 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     1141 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     1201 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     1261 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     1321 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     1381 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     1441 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     1501 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     1561 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     1621 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     1681 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     1741 gcgcacattt ccccgaaaag tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     1801 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     1861 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     1921 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattcgagct
     1981 cggtacctaa tgatagctag cgggccagta cccaaaaagc ggggggtctg gtggcagtgg
     2041 agggggatcc gtgagcaagg gcgaggagga taacgcctct ctcccagcga cacatgagtt
     2101 acacatcttt ggctccatca acggtgtgga ctttgacatg gtgggtcagg gcaccggcaa
     2161 tccaaatgat ggttatgagg agttaaacct gaagtccacc aagggtgacc tccagttctc
     2221 cccctggatt ctggtccctc atatcgggta tggcttccat cagtacctgc cctaccctga
     2281 cgggatgtcg cctttccagg ccgccatggt agatggctcc ggataccaag tccatcgcac
     2341 aatgcagttt gaagatggtg cctcccttac tgttaactac cgctacacct acgagggaag
     2401 ccacatcaaa ggagaggccc aggtgaaggg gactggtttc cctgctgacg gtcctgtgat
     2461 gaccaactcg ctgaccgctg cggactggtg caggtcgaag aagacttacc ccaacgacaa
     2521 aaccatcatc agtaccttta agtggagtta caccactgga aatggcaagc gctaccggag
     2581 cactgcgcgg accacctaca cctttgccaa gccaatggcg gctaactatc tgaagaacca
     2641 gccgatgtac gtgttccgta agacggagct caagcactcc aagaccgagc tcaacttcaa
     2701 ggagtggcaa aaggccttta ccgatgtgat gggcatggac gagctgtaca agtaagcggc
     2761 cgccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2821 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2881 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2941 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3001 atggacgcgt cagcaaaagg ccaggaaccg taaaaagggt aggggaggcg cttttcccaa
     3061 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
     3121 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg
     3181 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     3241 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     3301 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     3361 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
     3421 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc
     3481 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacca
     3541 attcccaata ctatcgtcga gtgagccgcc accatggtga gcaagggcga ggaggataac
     3601 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc
     3661 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     3721 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag
     3781 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     3841 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     3901 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     3961 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     4021 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg
     4081 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag
     4141 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac
     4201 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc
     4261 ggcatggacg agctgtacaa gggtaaaaag aagaaaaaga agtcaaagac aaagtgtgta
     4321 attatgtagt aggcggccgc cgctgatcag cctcgactgt gccttctagt tgccagccat
     4381 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     4441 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     4501 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     4561 gggatgcggt gggctctatg gacgcgtcag caaaaggcca ggaaccgtaa aaa
//
