LOCUS       sequence-232642-        5191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with YFP-LID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5191)
  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 5191)
  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-P25
FEATURES             Location/Qualifiers
     source          1..5191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             join(81..83,84..794)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(123..144)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(384..403)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     731..752
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             801..1121
                     /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    1188..1309
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1225..1244)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1279..1298
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1316..1349)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1356..1672
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1523..1658
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2294..2518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2525..2773
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2525..2545
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2696..2715
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2775..2808)
                     /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(2845..2864)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2846..2864)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2951..2968)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3122..3710)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3202..3221)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3881..4672,4673..4741))
                     /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     4504..4523
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4742..4846)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4914..4932
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4970..4992)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5092..5111
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      121 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      181 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      241 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct
      301 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      361 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
      421 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      481 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
      541 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
      601 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
      661 tgctgcccga caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga
      721 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
      781 acgagctgta caaggaattc ggagtgcagg tggaaaccat ctccccagga gacgggcgca
      841 ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt gaagatggaa
      901 agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta ggcaagcagg
      961 aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag agagccaaac
     1021 tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc atcccaccac
     1081 atgccactct cgtcttcgat gtggagcttc taaaactgga aacgcgtgga gtggaggaag
     1141 tggcggaggg ggtagttctc ttaaggcggc gaggtaatta gactagtaac ttgtttattg
     1201 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     1261 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag tcgatataac
     1321 ttcgtatagc atacattata cgaagttatg tcgacggtgt ggaaagtccc caggctcccc
     1381 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     1441 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     1501 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     1561 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     1621 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagaattcgc
     1681 caccatgact gagtacaagc ctactgtgcg tttagccacc agggatgatg tgccacgagc
     1741 cgtgaggaca ctagcagccg cctttgctga ctacccagct acaaggcaca ccgtggaccc
     1801 tgacagacac attgaaagag tcacagaact tcaggagctg tttttaacta gggtgggcct
     1861 ggatattggc aaagtctggg tggccgatga tggagcagca gttgcagtgt ggaccacccc
     1921 agagtctgtt gaagctggag ctgtctttgc agaaattgga ccaagaatgg ccgagttatc
     1981 tggcagcagg ctggctgctc aacagcagat ggaaggatta ttagctcccc acagacccaa
     2041 ggagcctgcc tggttcctgg caacagttgg agtttctcca gaccatcagg gcaagggctt
     2101 aggatctgct gtggtgctgc ctggtgtgga agcagctgag agagctgggg tgccagcatt
     2161 tctggagaca tcagctccta gaaacctgcc cttctatgaa cggctgggct tcacagtgac
     2221 tgctgatgtg gaggtgcctg agggccccag aacctggtgc atgacaagaa aaccaggagc
     2281 ctgactgcag cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2341 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2401 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2461 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2521 cgaggagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     2581 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     2641 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     2701 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     2761 aggacgaaac accgataact tcgtatagca tacattatac gaagttatgg taccgatgca
     2821 gctagcccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg
     2881 ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2941 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3001 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3061 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3121 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3181 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3241 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3301 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3361 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3421 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3481 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3541 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3601 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3661 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3721 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3781 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3841 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3901 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3961 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4021 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4081 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4141 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4201 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4261 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4321 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4381 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4441 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4501 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4561 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4621 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4681 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4741 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4801 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4861 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4921 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4981 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     5041 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     5101 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt
     5161 aatacataac cttatgtatc atacacatac g
//
