LOCUS       sequence-274800-        3521 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for PCR-generated donor DNA for rapid tagging of proteins 
            with GFP11-3xFlag-tagRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3521)
  AUTHORS   Paix A, Rasoloson D, Folkmann A, Seydoux G
  TITLE     Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome
            Engineering using Double-Stranded DNA Donors.
  JOURNAL   Curr Protoc Mol Biol. 2019 Dec;129(1):e102. doi: 10.1002/cpmb.102.
  PUBMED    31710422
REFERENCE   2  (bases 1 to 3521)
  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: "Curr Protoc
            Mol Biol."; date: "2019-12"; volume: "129(1):e102. doi"; pages: " 
            10.1002/cpmb.102"
COMMENT     Sequence Label: pAP3041
FEATURES             Location/Qualifiers
     source          1..3521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..48
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             58..123
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(879..895)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(879..895)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(892..914)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    903..919
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(927..933,934..951,952..957))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    972..993
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1110..1127)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1281..1869)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1361..1380)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2040..2831,2832..2900))
                     /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     2663..2682
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2901..3005)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3073..3091
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3129..3151)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3251..3270
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3464..3486
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3478..3495
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3479..3495
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cgtgaccaca tggtccttca tgagtatgta aatgctgctg ggattacagg tggcggcgac
       61 tacaaagatc acgacggcga ttacaaggac catgacatag attacaagga cgatgatgac
      121 aaggtatcta aaggggaaga actcatcaag gagaatatgc acatgaagct ctatatggaa
      181 ggtacagtaa acaatcacca cttcaaatgc acttctgaag gtgaaggaaa gccctacgag
      241 ggtacacaga cgatgagaat taaagtcgtt gagggaggcc cgctgccctt tgcttttgac
      301 atccttgcaa cctcatttat gtatggcagc cgaacattca tcaatcatac acaggggatt
      361 ccagattttt tcaaacagtc ctttccggag ggctttacat gggagcgagt aacgacctat
      421 gaagatgggg gagttttgac cgccacgcaa gatacgtcac tccaagacgg ctgcctgatt
      481 tacaatgtaa aaataagagg cgttaatttt ccgtctaatg gcccggtaat gcagaaaaag
      541 accctgggtt gggaggccaa cacggaaatg ctttatccgg cagacggtgg cttggagggg
      601 cgctctgaca tggcactgaa gttggtcggc ggaggtcacc ttatttgcaa cttcaaaact
      661 acgtaccgct caaagaagcc agccaaaaac cttaaaatgc ccggggttta ttatgtagac
      721 caccgcctcg aacggatcaa ggaagctgat aaagaaacgt atgtagagca acatgaagtc
      781 gcagtggcaa ggtactgtga tctccccagt aagctggggc acaaactgaa tgatcctcta
      841 gagtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga
      901 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
      961 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     1021 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1081 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     1141 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     1201 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1261 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1321 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1381 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1441 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1501 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1561 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1621 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1681 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     1741 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1801 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1861 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1921 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1981 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     2041 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     2101 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     2161 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     2221 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     2281 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     2341 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     2401 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     2461 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     2521 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     2581 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     2641 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     2701 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     2761 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     2821 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     2881 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2941 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     3001 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     3061 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     3121 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     3181 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     3241 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     3301 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc
     3361 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     3421 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     3481 aaaacgacgg ccagtgaatt cgagctcggt acccggggat c
//
