LOCUS       sequence-328191-        3419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector backbone to support a knock-in of 
            Linker-mScarlet-P2A-Puro at the C-terminus of a target locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3419)
  AUTHORS   Bollen Y, Hageman JH, van Leenen P, Derks LLM, Ponsioen B, Buissant 
            des Amorie JR, Verlaan-Klink I, van den Bos M, Terstappen LWMM, van 
            Boxtel R, Snippert HJG
  TITLE     Efficient and error-free fluorescent gene tagging in human organoids
            without double-strand DNA cleavage.
  JOURNAL   PLoS Biol. 2022 Jan 28;20(1):e3001527. doi: 
            10.1371/journal.pbio.3001527.
  PUBMED    35089911
REFERENCE   2  (bases 1 to 3419)
  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: "PLoS 
            Biol."; date: "2022-01-28"; volume: "20(1):e3001527. doi"; pages: " 
            10.1371/journal.pbio.3001527"
COMMENT     Sequence Label: TVBB C-term-mScarlet-Puro
FEATURES             Location/Qualifiers
     source          1..3419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..693
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(131..149)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     399..418
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             703..759
                     /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(760..779)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(1397..1414)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1568..2156)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1648..1667)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2327..3118,3119..3187))
                     /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     2950..2969
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3188..3292)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtgagcaagg gcgaggcagt gatcaaggag ttcatgagat tcaaggtgca catggagggc
       61 tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggcagacc ctacgagggc
      121 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttctc ctgggacatc
      181 ctgtcccctc agttcatgta cggctccagg gccttcacca agcaccccgc cgacatcccc
      241 gactactata agcagtcctt ccccgagggc ttcaagtggg agagagtgat gaacttcgag
      301 gacggcggcg ccgtgaccgt gacccaggac acctccctgg aggacggcac cctgatctac
      361 aaggtgaagc tcagaggcac caacttccct cctgacggcc ccgtaatgca gaagaagaca
      421 atgggctggg aagcatccac cgagagattg taccccgagg acggcgtgct gaagggcgac
      481 attaagatgg ccctgagact gaaggacggc ggccgctacc tggcggactt caagaccacc
      541 tacaaggcca agaagcccgt gcagatgccc ggcgcctaca acgtcgacag gaagttggac
      601 atcacctccc acaacgagga ctacaccgtg gtggaacagt acgaacgctc cgagggcaga
      661 cactccaccg gcggcatgga cgagctgtac aagggaagcg gagcgacgaa ttttagtcta
      721 ctgaaacaag cgggagacgt ggaggaaaac cctggaccta tgaccgagta caagcccacg
      781 gtgcgcctcg ccaccagaga cgacgtccct agagccgtaa gaaccctcgc cgccgctttt
      841 gccgactacc ctgccacaag acacaccgtc gatccggaca ggcacatcga gagagtcacc
      901 gagctgcaag aactcttcct cacgagggtc gggctcgaca tcggaaaggt gtgggtcgcg
      961 gacgacggag ccgcggtggc tgtctggacc acgcctgaga gtgtcgaagc aggggcagtg
     1021 ttcgccgaaa tcggcccaag aatggccgaa ttgtctggtt cccggctggc agctcagcaa
     1081 cagatggaag gcctcctggc gccacaccgg cctaaggagc ccgcgtggtt cctggctaca
     1141 gttggagttt caccagatca tcagggaaag ggtctgggct ctgctgtcgt gctcccagga
     1201 gtggaagctg ctgagagagc cggggtgcct gccttcctgg agacctctgc gcctaggaat
     1261 ctccccttct acgagagact cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     1321 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gaagagcgtt taaacgcgaa
     1381 ccggctcttc gaattcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     1441 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     1501 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1561 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1621 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1681 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1741 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1801 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     1861 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     1921 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     1981 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2041 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2101 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2161 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2221 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2281 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2341 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2401 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2461 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2521 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2581 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2641 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2701 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2761 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     2821 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     2881 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     2941 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3001 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3061 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3121 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3181 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3241 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     3301 cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     3361 cgctacggaa gagcgcgatc gcgtttaaag gctcttcagg cggaggcggc agcgccagc
//
