LOCUS       sequence-232613-        4807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4807)
  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 4807)
  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-P05
FEATURES             Location/Qualifiers
     source          1..4807
                     /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..797)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(81..83,84..794)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     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"
     polyA_signal    804..925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(841..860)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     895..914
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(932..965)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        972..1288
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1139..1274
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1910..2134
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2141..2389
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2141..2161
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2312..2331
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2391..2424)
                     /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(2461..2480)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2462..2480)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2567..2584)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2738..3326)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2818..2837)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3497..4288,4289..4357))
                     /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     4120..4139
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4358..4462)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4530..4548
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4586..4608)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4708..4727
                     /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 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
      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 ctgagcaccc agtccgccct gagcaaagac cccaacgaga
      721 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
      781 acgagctgta caagtaaact agtaacttgt ttattgcagc ttataatggt tacaaataaa
      841 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
      901 tgtccaaact catcaatgta tcttagtcga tataacttcg tatagcatac attatacgaa
      961 gttatgtcga cggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     1021 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     1081 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     1141 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     1201 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     1261 cttttttgga ggcctaggct tttgcaaaga attcgccacc atgactgagt acaagcctac
     1321 tgtgcgttta gccaccaggg atgatgtgcc acgagccgtg aggacactag cagccgcctt
     1381 tgctgactac ccagctacaa ggcacaccgt ggaccctgac agacacattg aaagagtcac
     1441 agaacttcag gagctgtttt taactagggt gggcctggat attggcaaag tctgggtggc
     1501 cgatgatgga gcagcagttg cagtgtggac caccccagag tctgttgaag ctggagctgt
     1561 ctttgcagaa attggaccaa gaatggccga gttatctggc agcaggctgg ctgctcaaca
     1621 gcagatggaa ggattattag ctccccacag acccaaggag cctgcctggt tcctggcaac
     1681 agttggagtt tctccagacc atcagggcaa gggcttagga tctgctgtgg tgctgcctgg
     1741 tgtggaagca gctgagagag ctggggtgcc agcatttctg gagacatcag ctcctagaaa
     1801 cctgcccttc tatgaacggc tgggcttcac agtgactgct gatgtggagg tgcctgaggg
     1861 ccccagaacc tggtgcatga caagaaaacc aggagcctga ctgcagcgac tgtgccttct
     1921 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     1981 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2041 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2101 agcaggcatg ctggggatgc ggtgggctct atggctcgag gagggcctat ttcccatgat
     2161 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
     2221 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     2281 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     2341 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg ataacttcgt
     2401 atagcataca ttatacgaag ttatggtacc gatgcagcta gcccgcggat ctgccggtct
     2461 ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc
     2521 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2581 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2641 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2701 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2761 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2821 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2881 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2941 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3001 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3061 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3121 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3181 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3241 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3301 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3361 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3421 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3481 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3541 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3601 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3661 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3721 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3781 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3841 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3901 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3961 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4021 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4081 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4141 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4201 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4261 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4321 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4381 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4441 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4501 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     4561 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     4621 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     4681 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4741 atatggacat attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac
     4801 acatacg
//
