LOCUS       sequence-328166-        2765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector backbone to support a knock-in of mNeongreen-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 2765)
  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 2765)
  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-mNeongreen
FEATURES             Location/Qualifiers
     source          1..2765
                     /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..1822
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      1370..1379
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1881..1898)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2052..2640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2132..2151)
                     /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 aggaggataa
     1141 catggcctct ctcccagcga cacatgagtt acacatcttt ggctctatca acggtgtgga
     1201 ctttgacatg gtgggtcagg gaaccggaaa tccaaatgat ggttatgagg agttaaatct
     1261 gaagtcaacc aagggtgacc tccagttctc tccttggatt ctggtccctc atatcgggta
     1321 tggcttccat cagtacctgc cttaccctga cgggatgtct cctttccagg ccgccatggt
     1381 agatggctct ggataccaag tccatagaac aatgcagttt gaagatggtg cctcccttac
     1441 tgttaattac agatacacat acgagggaag ccatatcaaa ggagaggccc aggtgaaggg
     1501 gactggtttc cctgctgacg gtcctgtgat gaccaactcg ctgaccgctg cggactggtg
     1561 caggtcgaag aaaacttacc ccaacgacaa aaccatcatc agtaccttta agtggagtta
     1621 caccactgga aatggcaaaa gataccggag cactgcgaga accacttaca cttttgccaa
     1681 gccaatggcg gctaactatc tgaagaacca gccgatgtac gtgttccgta agacggagct
     1741 caagcactcc aagaccgagc tcaacttcaa ggagtggcaa aaggccttta ccgatgtgat
     1801 gggcatggac gagctgtaca agggcggagg cggcagcgcc agccgaagag cgtttaaacg
     1861 cgaaccggct cttcgaattc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1921 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     1981 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2041 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2101 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2161 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2221 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     2281 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2341 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2401 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     2461 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     2521 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2581 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2641 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2701 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2761 tgaag
//
