LOCUS       sequence-232601-        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"
     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..1604
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1356..1376
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1527..1546
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1606..1639)
                     /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(1676..1695)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1677..1695)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1782..1799)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1953..2541)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2033..2052)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2712..3503,3504..3572))
                     /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     3335..3354
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3573..3677)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3745..3763
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3801..3823)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3923..3942
                     /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 tcgaggaggg cctatttccc atgattcctt
     1381 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     1441 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     1501 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     1561 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgataac ttcgtatagc
     1621 atacattata cgaagttatg gtaccgatgc agctagcccg cggatctgcc ggtctcccta
     1681 tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc
     1741 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     1801 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     1861 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     1921 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     1981 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2041 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2101 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2161 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2221 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2281 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     2341 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     2401 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     2461 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     2521 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     2581 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     2641 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     2701 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     2761 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2821 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     2881 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     2941 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3001 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3061 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3121 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3181 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3241 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     3301 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     3361 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3421 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3481 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     3541 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     3601 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     3661 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     3721 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     3781 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     3841 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     3901 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     3961 gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat catacacata
     4021 cg
//
