LOCUS       sequence-328204-        3440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector backbone to support a knock-in of 
            Linker-mTurquoise2-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 3440)
  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 3440)
  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-mTurquoise2-Puro
FEATURES             Location/Qualifiers
     source          1..3440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(39..830,831..899))
                     /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     662..681
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(900..1004)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1174..1195)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1435..1454)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1782..1803
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1855..1911
                     /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(1912..1931)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(2549..2566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2720..3308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2800..2819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
       61 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
      121 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
      181 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
      241 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
      301 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
      361 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
      421 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
      481 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
      541 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
      601 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
      661 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
      721 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
      781 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
      841 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
      901 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
      961 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     1021 gtgccacctg atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgctacgg
     1081 aagagcgcga tcgcgtttaa aggctcttca ggcggaggcg gcagcgccag cgtgagcaag
     1141 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaat
     1201 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1261 ctgaagttca tctgcaccac cggaaagctg cctgtgccct ggccaaccct cgtgaccacc
     1321 ctgtcttggg gcgtgcagtg cttcgccaga taccctgacc acatgaagca gcacgacttc
     1381 ttcaagtctg ccatgcccga aggctacgtc caggagagga ccatcttctt caaggacgac
     1441 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaacagaatc
     1501 gagctgaagg gcatcgactt caaggaggac ggaaacatcc tggggcacaa gctggagtac
     1561 aactacttct ctgacaacgt ctatatcacc gccgacaagc agaagaatgg catcaaggcc
     1621 aacttcaaga tccgccacaa catcgaggat ggcggagtgc agctcgccga tcactaccag
     1681 cagaataccc ctatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1741 cagtccaagc tgagcaaaga ccccaacgag aagagagatc acatggtcct gctggagttc
     1801 gtgaccgccg ccgggatcac tctcggaatg gacgagctgt acaagggaag cggagcgacg
     1861 aattttagtc tactgaaaca agcgggagac gtggaggaaa accctggacc tatgaccgag
     1921 tacaagccca cggtgcgcct cgccaccaga gacgacgtcc ctagagccgt aagaaccctc
     1981 gccgccgctt ttgccgacta ccctgccaca agacacaccg tcgatccgga caggcacatc
     2041 gagagagtca ccgagctgca agaactcttc ctcacgaggg tcgggctcga catcggaaag
     2101 gtgtgggtcg cggacgacgg agccgcggtg gctgtctgga ccacgcctga gagtgtcgaa
     2161 gcaggggcag tgttcgccga aatcggccca agaatggccg aattgtctgg ttcccggctg
     2221 gcagctcagc aacagatgga aggcctcctg gcgccacacc ggcctaagga gcccgcgtgg
     2281 ttcctggcta cagttggagt ttcaccagat catcagggaa agggtctggg ctctgctgtc
     2341 gtgctcccag gagtggaagc tgctgagaga gccggggtgc ctgccttcct ggagacctct
     2401 gcgcctagga atctcccctt ctacgagaga ctcggcttca ccgtcaccgc cgacgtcgag
     2461 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgaagagcg
     2521 tttaaacgcg aaccggctct tcgaattcct cgctcactga ctcgctgcgc tcggtcgttc
     2581 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2641 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2701 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2761 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2821 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2881 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2941 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3001 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3061 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3121 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3181 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3241 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3301 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3361 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3421 attaaaaatg aagttttaaa
//
