LOCUS       sequence-332664-        2753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector backbone to support a knock-in of mScarlet-Linker 
            at the N-terminus of a target locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2753)
  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 2753)
  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 N-term-mScarlet
FEATURES             Location/Qualifiers
     source          1..2753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(46..837,838..906))
                     /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     669..688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(907..1011)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1118..1810
                     /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(1248..1266)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1516..1535
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1869..1886)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2040..2628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2120..2139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
       61 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
      121 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
      181 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
      241 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
      301 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
      361 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
      421 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
      481 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
      541 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
      601 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
      661 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
      721 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
      781 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
      841 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
      901 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
      961 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     1021 ccgaaaagtg ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     1081 gctacggaag agcgcgatcg cgtttaaagg ctcttcagtg agcaagggcg aggcagtgat
     1141 caaggagttc atgagattca aggtgcacat ggagggctcc atgaacggcc acgagttcga
     1201 gatcgagggc gagggcgagg gcagacccta cgagggcacc cagaccgcca agctgaaggt
     1261 gaccaagggt ggccccctgc ccttctcctg ggacatcctg tcccctcagt tcatgtacgg
     1321 ctccagggcc ttcaccaagc accccgccga catccccgac tactataagc agtccttccc
     1381 cgagggcttc aagtgggaga gagtgatgaa cttcgaggac ggcggcgccg tgaccgtgac
     1441 ccaggacacc tccctggagg acggcaccct gatctacaag gtgaagctca gaggcaccaa
     1501 cttccctcct gacggccccg taatgcagaa gaagacaatg ggctgggaag catccaccga
     1561 gagattgtac cccgaggacg gcgtgctgaa gggcgacatt aagatggccc tgagactgaa
     1621 ggacggcggc cgctacctgg cggacttcaa gaccacctac aaggccaaga agcccgtgca
     1681 gatgcccggc gcctacaacg tcgacaggaa gttggacatc acctcccaca acgaggacta
     1741 caccgtggtg gaacagtacg aacgctccga gggcagacac tccaccggcg gcatggacga
     1801 gctgtacaag ggcggaggcg gcagcgccag ccgaagagcg tttaaacgcg aaccggctct
     1861 tcgaattcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1921 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1981 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2041 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2101 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2161 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2221 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2281 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2341 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2401 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2461 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2521 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2581 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2641 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2701 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aag
//
