LOCUS       sequence-343517-        5540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology Directed Repair construct for C-terminal tagging of mMPP8 
            with mAID using CRISPR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5540)
  AUTHORS   Muller I, Moroni AS, Shlyueva D, Sahadevan S, Schoof EM, 
            Radzisheuskaya A, Hojfeldt JW, Tatar T, Koche RP, Huang C, Helin K
  TITLE     MPP8 is essential for sustaining self-renewal of ground-state 
            pluripotent stem cells.
  JOURNAL   Nat Commun. 2021 May 24;12(1):3034. doi: 10.1038/s41467-021-23308-4.
  PUBMED    34031396
REFERENCE   2  (bases 1 to 5540)
  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: "Nat 
            Commun."; date: "2021-05-24"; volume: "12(1):3034. doi"; pages: " 
            10.1038/s41467-021-23308-4"
COMMENT     Sequence Label: pRepair-MPP8_mAID-P2A-BFP
FEATURES             Location/Qualifiers
     source          1..5540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..38)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(22..38)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(35..57)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    46..62
                     /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(70..76,77..94,95..100))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    115..136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(253..270)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(424..1012)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(504..523)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1183..1974,1975..2043))
                     /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     1806..1825
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2044..2148)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2216..2234
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2272..2294)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2394..2413
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2607..2629
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2621..2638
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2622..2638
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3610..3813
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             3823..3879
                     /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             3880..4587
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
ORIGIN
        1 atcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
       61 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
      121 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
      181 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
      241 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
      301 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      361 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      421 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      481 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      541 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      601 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      661 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      721 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      781 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      841 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      901 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      961 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     1021 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     1081 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     1141 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     1201 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1261 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1321 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1381 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1441 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1501 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1561 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1621 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1681 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1741 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1801 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1861 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1921 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1981 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     2041 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     2101 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     2161 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     2221 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     2281 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     2341 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     2401 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     2461 gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg
     2521 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     2581 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     2641 attcgatgta tattagtgaa cttcagacaa tccccactca ctataaaaga aaatgatcca
     2701 aattgttaga acatgttaat gtttattcac ttaaatcgtg ctaccatatg tcctacagaa
     2761 aacaatgtga gactagaata gaatagtagc atgccacgcc ctcattctaa cacactctgc
     2821 tgctggaact cttccagagg ctaataaaat gaacaccttg tattgaatgc agtcatgtgt
     2881 tgtttttatt tcagccctag gtagcctaga actctttaga catggtaagc cttgaaggtg
     2941 aggtttttgg ggggctgcac agagagatcc tatcttaaaa ttcaaaaaca aaacccctca
     3001 gttcgctgat cggagttcct ggtctctatt gtaaatgtca cagccctgac ttgagtctga
     3061 ctttatcttc tctctttccc ctctaggaca gtcactttgt ttattcattc agtcctgttg
     3121 ctggccccaa taagttgttt ataaggttga cggaagcacc ctttgccaag gtgaaaatct
     3181 ttgcttcata tttacacagt aactgctgac ataataatag caatactaat aaatcctctc
     3241 actagaaacg tcaggagaca tggttcagac aagctctttg tactagacgg cttatgttgg
     3301 gcaaacttcg gctctctgag gcagctatgc atagcatatt tggcaagtct gggggcatcc
     3361 ctgctgagct ctccaaatct tgacttcagt actaaaatgc cactaccctc acctaggaca
     3421 gatccctcca tatgaaaaaa ggaagctgga ctacttagaa gaccattcta ataatggtta
     3481 cctaatatat attatagcaa taacttggat gttcttacaa ggtggaattt gtgtactgac
     3541 attcattctc ttctttaggt taagttgcta ataggtgcat acagagtaca gctgcagggt
     3601 ggttctggta aggagaagag tgcttgtcct aaagatccag ccaaacctcc ggccaaggca
     3661 caagttgtgg gatggccacc ggtgagatca taccggaaga acgtgatggt ttcctgccaa
     3721 aaatcaagcg gtggcccgga ggcggcggcg ttcgtgaagg tatcaatgga cggagcaccg
     3781 tacttgagga aaatcgattt gaggatgtat aaaggatccg gcgcaacaaa cttctctctg
     3841 ctgaaacaag ccggagatgt cgaagagaat cctggaccgg tgtctaaggg cgaagagctg
     3901 attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc
     3961 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
     4021 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac
     4081 ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc
     4141 cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct
     4201 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
     4261 aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc
     4321 gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc
     4381 gtgggcggga gccatctgat cgcaaacgcc aagaccacat atagatccaa gaaacccgct
     4441 aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag
     4501 gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc
     4561 cctagcaaac tggggcacaa gcttaattaa ccacaaagga tgcagaaccg gcctcttttc
     4621 agctggattc caaggaccat ccctgtagca gtttggaatt ctggtgcagc tcaaatgttc
     4681 ctacctagtt gtaaacttaa aatttaagcc tgctgcctgt tcaggtttgt ctacatatat
     4741 ttgtgccatt ggtcgaaaac gttcaatgtt ctgcttagtt tatttccaca atgtggaaca
     4801 gtacgtgtaa gaaatattta aggaaattaa aaaccttttg aattgaaatt ttctactagt
     4861 cgttgtgagt ttgtatttga aaaacaaaaa ccagattcag ggtgggcccc actactgagt
     4921 tcaagagtag ccatgtctac aacaacatga accttttaca aatcaggttt aaacatttac
     4981 tatacaacta agtttgatat tcttaaaggt ggaaaagcaa cttaattata attataaaat
     5041 gtaacaatgc agctgcagga ttttcctctg agcctctttg tttccagaat aaagacatct
     5101 gagattgaat tataagcttg ggcttgttct gactcgcttg taacttaacc aacacgttta
     5161 ttctattcca tggtcagagg caattctgtc ccctcacccg attatcccag agtcctcttc
     5221 cctaccagaa cttccaccta tttccttcct gcctcagcta gcaggccatc agctacttat
     5281 ttatggcagg tgatgcttcc atacagtaca caacagattc tctctacagt agaaatgtgt
     5341 cccgtagcat actcttgaca tgtgctgtga ctgctcataa gcataaatca caaaataagg
     5401 ttttctgcct gcggcttttg cgtcattagg agtttttcct cacagcgtgc tgtaacctgt
     5461 aagagacaaa gctttcatca ataaatggga atggtataca tgtgcacaca tgcatgcatt
     5521 ttgtcctttg taacacagcc
//
