LOCUS       sequence-351918-        7548 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For genome editing: EGFP-inducible Caspase9 knock-in cassette which 
            allows ablation of target cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7548)
  AUTHORS   Okamoto T, Natsume Y, Yamanaka H, Fukuda M, Yao R
  TITLE     A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal
            cancer patient-derived organoids.
  JOURNAL   STAR Protoc. 2021 Sep 16;2(4):100780. doi: 
            10.1016/j.xpro.2021.100780. eCollection 2021 Dec 17.
  PUBMED    34585151
REFERENCE   2  (bases 1 to 7548)
  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: "STAR 
            Protoc."; date: "2021-09-16"; volume: "2(4):100780. doi"; pages: " 
            10.1016/j.xpro.2021.100780. eCollection 2021 Dec 17"
COMMENT     Sequence Label: pCMMP-MCS-IRES-eGFP-P2A-iCas9
FEATURES             Location/Qualifiers
     source          1..7548
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             371..787
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     717..739
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     755..771
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1046..1612
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1062..1614
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1206..1223)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1433..1452
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1626..1628,1629..1631,1632..2342)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1671..1692)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1932..1951)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2279..2300
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2361..2417
                     /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             2430..2750
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             complement(3102..3113)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             3627..3653
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4929..4945)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4929..4945)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4942..4964)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4953..4969
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4977..4983,4984..5001,5002..5007))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5022..5043
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5160..5177)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5331..5919)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5411..5430)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6090..6881,6882..6950))
                     /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     6713..6732
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6951..7055)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7123..7141
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7179..7201)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7301..7320
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7514..7536
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7528..7545
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7529..7545
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aagcttgcat gcctgcaggt cgactagtta gctaactagc tctgtatctg gcggacccgt
       61 ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc cagggacttc
      121 gggggccgtt tttgtggccc gacctgagtc ctaaaatccc gatcgtttag gactctttgg
      181 tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc taaaacagtt
      241 cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg cgcgtcttgt
      301 ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt tgtctgaaaa
      361 tatgggcccg ggctagactg ttaccactcc cttaagtttg accttaggtc actggaaaga
      421 tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt gggttacctt
      481 ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca cctttaaccg
      541 agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg gacacccaga
      601 ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc cctgggtcaa
      661 gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt ctctccccct
      721 tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca ctccttctct
      781 aggcgccccc atatggccat atgagatctt atatggggca cccccgcccc ttgtaaactt
      841 ccctgaccct gacatgacaa gagttactaa cagcccctct ctccaagctc acttacaggc
      901 tctctactta gtccagcacg aagtctggag acctctggcg gcagcctacc aagaacaact
      961 ggaccgaccg gtcaattggt taacgctgag cttcgaagcc ggcgcggccg cacgcgtatg
     1021 catgtatacc tcgaggatcc ggaattccgc ccccccccct aacgttactg gccgaagccg
     1081 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt
     1141 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct
     1201 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct
     1261 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
     1321 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc
     1381 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc
     1441 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc
     1501 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct
     1561 aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca
     1621 caaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1681 tggacggcga cgtaaacggc cacaagttca gcgtgtctgg cgagggcgag ggcgatgcca
     1741 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1801 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1861 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1921 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1981 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2041 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2101 agaacggcat caaggcgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2161 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2221 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2281 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2341 agttaatcgc cggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg
     2401 aggagaaccc tggacctacc atgctcgagg gagtgcaggt ggagactatc tccccaggag
     2461 acgggcgcac cttccccaag cgcggccaga cctgcgtggt gcactacacc gggatgcttg
     2521 aagatggaaa gaaagttgat tcctcccggg acagaaacaa gccctttaag tttatgctag
     2581 gcaagcagga ggtgatccga ggctgggaag aaggggttgc ccagatgagt gtgggtcaga
     2641 gagccaaact gactatatct ccagattatg cctatggtgc cactgggcac ccaggcatca
     2701 tcccaccaca tgccactctc gtcttcgatg tggagcttct aaaactggaa tctggcggtg
     2761 gatccggagt cgacggattt ggtgatgtcg gtgctcttga gagtttgagg ggaaatgcag
     2821 atttggctta catcctgagc atggagccct gtggccactg cctcattatc aacaatgtga
     2881 acttctgccg tgagtccggg ctccgcaccc gcactggctc caacatcgac tgtgagaagt
     2941 tgcggcgtcg cttctcctcg ctgcatttca tggtggaggt gaagggcgac ctgactgcca
     3001 agaaaatggt gctggctttg ctggagctgg cgcggcagga ccacggtgct ctggactgct
     3061 gcgtggtggt cattctctct cacggctgtc aggccagcca cctgcagttc ccaggggctg
     3121 tctacggcac agatggatgc cctgtgtcgg tcgagaagat tgtgaacatc ttcaatggga
     3181 ccagctgccc cagcctggga gggaagccca agctcttttt catccaggcc tgtggtgggg
     3241 agcagaaaga ccatgggttt gaggtggcct ccacttcccc tgaagacgag tcccctggca
     3301 gtaaccccga gccagatgcc accccgttcc aggaaggttt gaggaccttc gaccagctgg
     3361 acgccatatc tagtttgccc acacccagtg acatctttgt gtcctactct actttcccag
     3421 gttttgtttc ctggagggac cccaagagtg gctcctggta cgttgagacc ctggacgaca
     3481 tctttgagca gtgggctcac tctgaagacc tgcagtccct cctgcttagg gtcgctaatg
     3541 ctgtttcggt gaaagggatt tataaacaga tgcctggttg ctttaatttc ctccggaaaa
     3601 aacttttctt taaaacatca gtcgactatc cgtacgacgt accagactac gcataattaa
     3661 ttaagaattc cggattagtc caatttgtta aagacaggat atcagtggtc caggctctag
     3721 ttttgactca acaatatcac cagctgaagc ctatagagta cgagccatag ataaaataaa
     3781 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca
     3841 agctagctta agtaacgcca ttttgcaagg catggaaaat acataactga gaatagagaa
     3901 gttcagatca aggttaggaa cagagagaca gcagaatatg ggccaaacag gatatctgtg
     3961 gtaagcagtt cctgccccgg ctcagggcca agaacagttg gaacagcaga atatgggcca
     4021 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc
     4081 cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc
     4141 aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc
     4201 tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggcgc
     4261 gccagtcctc cgatagactg cgtcgcccgg gtacccgtgt tctcaataaa ccctcttgca
     4321 gttgcatccg actcgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta
     4381 cccgtcagcg ggggtctttc agtttctccc acctacacag gtctcactaa cattcctgat
     4441 gtgccgcagg gactccgtca gcccggtttt tgtttataat aaaatgcaag aacagtgttc
     4501 ccttcaagcc agactacatc ctgactctcg gctttataaa agaatgttga agggctctgt
     4561 ggactatctg ccacacgact tttaagattt ttatgcctcc tggatgaggg atttagtcaa
     4621 tctatcctcg tctattttgc tggcttctcc gtattttaaa tttctagttt gcactccctt
     4681 cctgagagca cggcgattgc agagtagtta atactctgag ggcaggcttc tgtgaaaagg
     4741 ttgcctgggc tcagtgtgag attttgccat aaaaaggggt cctgcccctg tgtacagaca
     4801 gatcggaatc tagagtgcat actcagagtc cccgcggttc cggggctctg atctcagggc
     4861 atctttgcct agagatcctc tacgccggac gcatcgtggc cgggtaccga gctcgaattc
     4921 gtaatcatgt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4981 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5041 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5101 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5161 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5221 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5281 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5341 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5401 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5461 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5521 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5581 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5641 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5701 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5761 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5821 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5881 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5941 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6001 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6061 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6121 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6181 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6241 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6301 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6361 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6421 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6481 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6541 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6601 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6661 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6721 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6781 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6841 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6901 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6961 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7021 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7081 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     7141 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     7201 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     7261 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     7321 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     7381 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     7441 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     7501 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgcc
//
