LOCUS       sequence-328160-        3227 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector backbone to support a knock-in of 
            Linker-mNeongreen-P2A-Blast at the C-terminus of a target locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3227)
  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 3227)
  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-mNeongreen-Blast
FEATURES             Location/Qualifiers
     source          1..3227
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     399..418
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(637..741)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             869..1573
                     /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      1121..1130
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1583..1639
                     /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"
     CDS             1640..2035
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(2073..2090)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2244..2832)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2324..2343)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3003..3227,1..567,568..636))
                     /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"
ORIGIN
        1 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
       61 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
      121 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
      181 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
      241 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
      301 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
      361 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
      421 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
      481 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
      541 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
      601 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
      661 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
      721 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgatg cggtgtgaaa
      781 taccgcacag atgcgtaagg agaaaatacc gctacggaag agcgcgatcg cgtttaaagg
      841 ctcttcaggc ggaggcggca gcgccagcgt gagcaagggc gaggaggata acatggcctc
      901 tctcccagcg acacatgagt tacacatctt tggctctatc aacggtgtgg actttgacat
      961 ggtgggtcag ggaaccggaa atccaaatga tggttatgag gagttaaatc tgaagtcaac
     1021 caagggtgac ctccagttct ctccttggat tctggtccct catatcgggt atggcttcca
     1081 tcagtacctg ccttaccctg acgggatgtc tcctttccag gccgccatgg tagatggctc
     1141 tggataccaa gtccatagaa caatgcagtt tgaagatggt gcctccctta ctgttaatta
     1201 cagatacaca tacgagggaa gccatatcaa aggagaggcc caggtgaagg ggactggttt
     1261 ccctgctgac ggtcctgtga tgaccaactc gctgaccgct gcggactggt gcaggtcgaa
     1321 gaaaacttac cccaacgaca aaaccatcat cagtaccttt aagtggagtt acaccactgg
     1381 aaatggcaaa agataccgga gcactgcgag aaccacttac acttttgcca agccaatggc
     1441 ggctaactat ctgaagaacc agccgatgta cgtgttccgt aagacggagc tcaagcactc
     1501 caagaccgag ctcaacttca aggagtggca aaaggccttt accgatgtga tgggcatgga
     1561 cgagctgtac aagggaagcg gagcgacgaa ttttagtcta ctgaaacaag cgggagacgt
     1621 ggaggaaaac cctggacctg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag
     1681 agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc
     1741 tctctctagc gacggcagga tcttcactgg tgtcaatgta tatcatttta ctgggggacc
     1801 ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg
     1861 tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggaa gatgccgaca
     1921 ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc
     1981 gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctgacgaag
     2041 agcgtttaaa cgcgaaccgg ctcttcgaat tcctcgctca ctgactcgct gcgctcggtc
     2101 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     2161 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2221 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     2281 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2341 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2401 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     2461 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     2521 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2581 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2641 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     2701 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2761 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2821 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2881 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2941 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     3001 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     3061 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     3121 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     3181 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caacttt
//
