LOCUS       sequence-228030-        4022 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with YFP-LID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4022)
  AUTHORS   Fueller J, Herbst K, Meurer M, Gubicza K, Kurtulmus B, Knopf JD, 
            Kirrmaier D, Buchmuller BC, Pereira G, Lemberg MK, Knop M
  TITLE     CRISPR-Cas12a-assisted PCR tagging of mammalian genes.
  JOURNAL   J Cell Biol. 2020 Jun 1;219(6). pii: 151766. doi: 
            10.1083/jcb.201910210.
  PUBMED    32406907
REFERENCE   2  (bases 1 to 4022)
  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: "J Cell 
            Biol."; date: "2020-06-1"; volume: "219(6). pii: 151766. doi"; 
            pages: " 10.1083/jcb.201910210"
COMMENT     Sequence Label: pMaCTag-25
FEATURES             Location/Qualifiers
     source          1..4022
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(64..66,67..777)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(106..127)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(367..386)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     714..735
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             784..1104
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     polyA_signal    1171..1292
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1208..1227)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1262..1281
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1299..1332)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1339..1587
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1339..1359
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1510..1529
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1589..1622)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1659..1678)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1660..1678)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1765..1782)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1936..2524)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2016..2035)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2695..3486,3487..3555))
                     /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     3318..3337
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3556..3660)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3728..3746
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3784..3806)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3906..3925
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4006..4022,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4006..4022,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      121 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      181 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      241 ctcgtgacca ccttcggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag
      301 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      361 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      421 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      481 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      541 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      601 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      661 tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      721 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggaa
      781 ttcggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc
      841 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaagt tgattcctcc
      901 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg
      961 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat
     1021 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc
     1081 gatgtggagc ttctaaaact ggaaacgcgt ggagtggagg aagtggcgga gggggtagtt
     1141 ctcttaaggc ggcgaggtaa ttagactagt aacttgttta ttgcagctta taatggttac
     1201 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     1261 tgtggtttgt ccaaactcat caatgtatct tagtcgatat aacttcgtat agcatacatt
     1321 atacgaagtt atgtcgagga gggcctattt cccatgattc cttcatattt gcatatacga
     1381 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
     1441 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     1501 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     1561 tatatcttgt ggaaaggacg aaacaccgat aacttcgtat agcatacatt atacgaagtt
     1621 atggtaccga tgcagctagc ccgcggatct gccggtctcc ctatagtgag tcgtattaat
     1681 ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1741 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     1801 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     1861 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1921 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1981 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2041 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2101 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     2161 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2221 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2281 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2341 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     2401 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2461 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2521 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     2581 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     2641 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     2701 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     2761 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     2821 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     2881 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     2941 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3001 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3061 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3121 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3181 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3241 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     3301 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     3361 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     3421 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     3481 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     3541 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     3601 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     3661 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     3721 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     3781 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     3841 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     3901 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga
     3961 acgcggctac aattaataca taaccttatg tatcatacac atacgattta ggtgacacta
     4021 ta
//
