LOCUS       sequence-332667-        3218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector backbone to support a knock-in of 
            Linker-mScarlet-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 3218)
  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 3218)
  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-mScarlet-Blast
FEATURES             Location/Qualifiers
     source          1..3218
                     /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             1139..1831
                     /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(1269..1287)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1537..1556
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1841..1897
                     /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             1898..2296
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(2334..2351)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2505..3093)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2585..2604)
                     /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 gcgccagcgt
     1141 gagcaagggc gaggcagtga tcaaggagtt catgagattc aaggtgcaca tggagggctc
     1201 catgaacggc cacgagttcg agatcgaggg cgagggcgag ggcagaccct acgagggcac
     1261 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttctcct gggacatcct
     1321 gtcccctcag ttcatgtacg gctccagggc cttcaccaag caccccgccg acatccccga
     1381 ctactataag cagtccttcc ccgagggctt caagtgggag agagtgatga acttcgagga
     1441 cggcggcgcc gtgaccgtga cccaggacac ctccctggag gacggcaccc tgatctacaa
     1501 ggtgaagctc agaggcacca acttccctcc tgacggcccc gtaatgcaga agaagacaat
     1561 gggctgggaa gcatccaccg agagattgta ccccgaggac ggcgtgctga agggcgacat
     1621 taagatggcc ctgagactga aggacggcgg ccgctacctg gcggacttca agaccaccta
     1681 caaggccaag aagcccgtgc agatgcccgg cgcctacaac gtcgacagga agttggacat
     1741 cacctcccac aacgaggact acaccgtggt ggaacagtac gaacgctccg agggcagaca
     1801 ctccaccggc ggcatggacg agctgtacaa gggaagcgga gcgacgaatt ttagtctact
     1861 gaaacaagcg ggagacgtgg aggaaaaccc tggacctatg gccaagcctt tgtctcaaga
     1921 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
     1981 ctacagcgtc gccagcgcag ctctctctag cgacggcagg atcttcactg gtgtcaatgt
     2041 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
     2101 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
     2161 cccctgcgga agatgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt
     2221 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
     2281 tgtgtgggag ggctgacgaa gagcgtttaa acgcgaaccg gctcttcgaa ttcctcgctc
     2341 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2401 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2461 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2521 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2581 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2641 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2701 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2761 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2821 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2881 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2941 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3001 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3061 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3121 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3181 aggatcttca cctagatcct tttaaattaa aaatgaag
//
