LOCUS       sequence-227996-        3635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3635)
  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 3635)
  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-06
FEATURES             Location/Qualifiers
     source          1..3635
                     /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..1200
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     952..972
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1123..1142
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1202..1235)
                     /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(1272..1291)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1273..1291)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1378..1395)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1549..2137)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1629..1648)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2308..3099,3100..3168))
                     /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     2931..2950
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3169..3273)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3341..3359
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3397..3419)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3519..3538
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3619..3635,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3619..3635,1)
                     /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 ggagggccta
      961 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
     1021 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
     1081 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
     1141 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
     1201 gataacttcg tatagcatac attatacgaa gttatggtac cgatgcagct agcccgcgga
     1261 tctgccggtc tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta
     1321 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     1381 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1441 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1501 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1561 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1621 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1681 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1741 tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1801 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1861 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1921 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1981 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2041 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2101 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2161 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2221 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2281 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2341 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2401 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2461 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2521 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2581 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     2641 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2701 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2761 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     2821 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     2881 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     2941 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3001 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3061 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3121 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     3181 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3241 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     3301 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     3361 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3421 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3481 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     3541 gagagtgcac catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt
     3601 atgtatcata cacatacgat ttaggtgaca ctata
//
