LOCUS       sequence-228043-        4804 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4804)
  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 4804)
  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-P06
FEATURES             Location/Qualifiers
     source          1..4804
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    784..905
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(821..840)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     875..894
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(912..945)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        952..1268
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1119..1254
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1890..2114
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2121..2369
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2121..2141
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2292..2311
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2371..2404)
                     /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(2441..2460)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2442..2460)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2547..2564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2718..3306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2798..2817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3477..4268,4269..4337))
                     /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     4100..4119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4338..4442)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4510..4528
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4566..4588)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4688..4707
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4788..4804,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4788..4804,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccagcaagg gcgaggagct gttcaccggc gtggtgccca tcctggtgga gctggacggc
      121 gacgtgaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgacgc caccaacggc
      181 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctg
      241 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcgc
      301 cacgacttct tcaagagcgc catgcccgag ggctacgtgc aggagcgcac catcagcttc
      361 aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      421 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct gggccacaag
      481 ctggagtaca acttcaacag ccacaacgtg tacatcaccg ccgacaagca gaagaacggc
      541 atcaaggcca acttcaagat ccgccacaac gtggaggacg gcagcgtgca gctggccgac
      601 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      661 ctgagcaccc agagccgcct gagcaaggac cccaacgaga agcgcgacca catggtgctg
      721 ctggagttcg tgaccgccgc cggcatcacc cacggcatgg acgagctgta caagtgaact
      781 agtaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
      841 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
      901 tcttagtcga tataacttcg tatagcatac attatacgaa gttatgtcga cggtgtggaa
      961 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     1021 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     1081 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     1141 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     1201 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     1261 tttgcaaaga attcgccacc atgactgagt acaagcctac tgtgcgttta gccaccaggg
     1321 atgatgtgcc acgagccgtg aggacactag cagccgcctt tgctgactac ccagctacaa
     1381 ggcacaccgt ggaccctgac agacacattg aaagagtcac agaacttcag gagctgtttt
     1441 taactagggt gggcctggat attggcaaag tctgggtggc cgatgatgga gcagcagttg
     1501 cagtgtggac caccccagag tctgttgaag ctggagctgt ctttgcagaa attggaccaa
     1561 gaatggccga gttatctggc agcaggctgg ctgctcaaca gcagatggaa ggattattag
     1621 ctccccacag acccaaggag cctgcctggt tcctggcaac agttggagtt tctccagacc
     1681 atcagggcaa gggcttagga tctgctgtgg tgctgcctgg tgtggaagca gctgagagag
     1741 ctggggtgcc agcatttctg gagacatcag ctcctagaaa cctgcccttc tatgaacggc
     1801 tgggcttcac agtgactgct gatgtggagg tgcctgaggg ccccagaacc tggtgcatga
     1861 caagaaaacc aggagcctga ctgcagcgac tgtgccttct agttgccagc catctgttgt
     1921 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1981 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2041 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2101 ggtgggctct atggctcgag gagggcctat ttcccatgat tccttcatat ttgcatatac
     2161 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     2221 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2281 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2341 tatatatctt gtggaaagga cgaaacaccg ataacttcgt atagcataca ttatacgaag
     2401 ttatggtacc gatgcagcta gcccgcggat ctgccggtct ccctatagtg agtcgtatta
     2461 atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2521 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2581 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2641 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2701 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2761 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2821 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2881 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
     2941 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3001 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3061 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3121 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     3181 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3241 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3301 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3361 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3421 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3481 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3541 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3601 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3661 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3721 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3781 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3841 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3901 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3961 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4021 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4081 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4141 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4201 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4261 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4321 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4381 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4441 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     4501 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     4561 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     4621 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     4681 aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta
     4741 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
     4801 tata
//
