LOCUS       sequence-232677-        4966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with YFP-LID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4966)
  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 4966)
  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-Z25
FEATURES             Location/Qualifiers
     source          1..4966
                     /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"
     regulatory      1679..1688
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1685..2059
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    2069..2293
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2300..2548
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2300..2320
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2471..2490
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2550..2583)
                     /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(2620..2639)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2621..2639)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2726..2743)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2897..3485)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2977..2996)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3656..4447,4448..4516))
                     /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     4279..4298
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4517..4621)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4689..4707
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4745..4767)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4867..4886
                     /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 caccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc
     1741 ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc
     1801 cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc
     1861 ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc
     1921 ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga
     1981 gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt
     2041 ggccgaggag caggactgac tgcagcgact gtgccttcta gttgccagcc atctgttgtt
     2101 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     2161 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     2221 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     2281 gtgggctcta tggctcgagg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2341 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     2401 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2461 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2521 atatatcttg tggaaaggac gaaacaccga taacttcgta tagcatacat tatacgaagt
     2581 tatggtaccg atgcagctag cccgcggatc tgccggtctc cctatagtga gtcgtattaa
     2641 tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2701 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2761 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2821 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2881 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2941 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3001 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3061 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3121 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3181 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3241 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3301 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     3361 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3421 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3481 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3541 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3601 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3661 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3721 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3781 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3841 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3901 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3961 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4021 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4081 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4141 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4201 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4261 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4321 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     4381 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4441 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4501 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     4561 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     4621 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     4681 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     4741 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4801 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     4861 actatgcggc atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag
     4921 aacgcggcta caattaatac ataaccttat gtatcataca catacg
//
