LOCUS       sequence-332663-        3413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector backbone to support a knock-in of 
            Linker-Dendra2-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 3413)
  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 3413)
  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-Dendra2-Puro
FEATURES             Location/Qualifiers
     source          1..3413
                     /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             1835..1891
                     /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(1892..1911)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(2529..2546)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2700..3288)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2780..2799)
                     /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 ctcttcaggc ggaggcggca gcgccagcaa
     1141 caccccggga attaacctga tcaaggagga catgagagtg aaggtgcaca tggagggcaa
     1201 cgtgaacggc cacgccttcg tgatcgaggg agagggcaag ggtaagccat acgagggcac
     1261 ccagacagcc aacctgaccg tgaaggaggg cgcccccctg ccattcagct acgacatcct
     1321 gaccaccgcc gtgcactacg gcaacagagt gttcaccaag taccccgagg atatcccaga
     1381 ctactttaag cagagcttcc ccgagggcta cagctgggag cgcaccatga ccttcgagga
     1441 caagggcatc tgcaccatcc gcagcgacat cagcctggag ggagactgct tctttcagaa
     1501 cgtgagattc aagggcacaa acttccctcc caacggcccc gtgatgcaga agaagaccct
     1561 gaagtgggag cccagcacag agaagctgca cgtgagagat ggcctgctgg tgggcaacat
     1621 caacatggcc ctgctgctgg agggaggagg ccactacctg tgcgacttta agaccaccta
     1681 caaggccaag aaggtggtgc agctgccaga cgcccacttc gtggaccaca gaatcgagat
     1741 cctgggaaac gacagcgatt acaacaaggt gaagctgtac gagcacgccg tggccagata
     1801 cagccccctg cccagccagg tgtggggaag cggagcgacg aattttagtc tactgaaaca
     1861 agcgggagat gtggaggaaa accctggacc tatgaccgag tacaagccca cggtgcgcct
     1921 cgccaccaga gacgacgtcc ctagagccgt aagaaccctc gccgccgctt ttgccgacta
     1981 ccctgccaca agacacaccg tcgatccgga caggcacatc gagagagtca ccgagctgca
     2041 agaactcttc ctcacgaggg tcgggctcga catcggaaag gtgtgggtcg cggacgacgg
     2101 agccgcggtg gctgtctgga ccacgcctga gagtgtcgaa gcaggggcag tgttcgccga
     2161 aatcggccca agaatggccg aattgtctgg ttcccggctg gcagctcagc aacagatgga
     2221 aggcctcctg gcgccacacc ggcctaagga gcccgcgtgg ttcctggcta cagttggagt
     2281 ttcaccagat catcagggaa agggtctggg ctctgctgtc gtgctcccag gagtggaagc
     2341 tgctgagaga gccggggtgc ctgccttcct ggagacctct gcgcctagga atctcccctt
     2401 ctacgagaga ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     2461 ctggtgcatg acccgcaagc ccggtgcctg acgaagagcg tttaaacgcg aaccggctct
     2521 tcgaattcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2581 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2641 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2701 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2761 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2821 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2881 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2941 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3001 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3061 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3121 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3181 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3241 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3301 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3361 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aag
//
