LOCUS       sequence-228020-        3602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mEos4b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3602)
  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 3602)
  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-17
FEATURES             Location/Qualifiers
     source          1..3602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..744
                     /codon_start=1
                     /product="fixation-resistant truly monomeric green-to-red 
                     photoswitchable fluorescent protein (Paez-Segala et al., 
                     2015)"
                     /label=mEos4b
                     /note="derived from mEos2"
                     /translation="VSAIKPDMRIKLRMEGNVNGHHFVIDGDGTGKPYEGKQTMDLEVK
                     EGGPLPFAFDILTTAFHYGNRVFVKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNAR
                     NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLL
                     EGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLPD
                     NARR"
     polyA_signal    751..872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(788..807)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     842..861
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(879..912)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        919..1167
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     919..939
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1090..1109
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1169..1202)
                     /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(1239..1258)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1240..1258)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1345..1362)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1516..2104)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1596..1615)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2275..3066,3067..3135))
                     /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     2898..2917
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3136..3240)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3308..3326
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3364..3386)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3486..3505
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3586..3602,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3586..3602,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagtg cgattaagcc agacatgagg atcaaactcc gtatggaagg caacgtaaac
      121 gggcaccact ttgtgatcga cggagatggt acaggcaagc cttatgaggg aaaacagacc
      181 atggatcttg aagtcaaaga gggcggacct ctgccttttg cctttgatat cctgaccact
      241 gcattccatt acggcaacag ggtattcgtg aaatatccag acaacataca agactatttt
      301 aagcagtcgt ttcctaaggg gtattcgtgg gaacgaagct tgactttcga agacgggggc
      361 atttgcaatg ccagaaacga cataacaatg gaaggggaca ctttctataa taaagttcga
      421 ttttatggta ccaactttcc cgccaatggt ccagttatgc agaagaagac gctgaaatgg
      481 gagccctcca ctgagaaaat gtatgtgcgt gatggagtgc tgacgggtga tattgagatg
      541 gctttgttgc ttgaaggaaa tgcccattac cgatgtgact tcagaactac ttacaaagct
      601 aaggagaagg gtgtcaagtt accaggcgcc cactttgtgg accacgccat tgagatttta
      661 agccatgaca aagattacaa caaggttaag ctgtatgagc atgctgttgc tcattctgga
      721 ttgcctgaca atgccagacg atgaactagt aacttgttta ttgcagctta taatggttac
      781 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
      841 tgtggtttgt ccaaactcat caatgtatct tagtcgatat aacttcgtat agcatacatt
      901 atacgaagtt atgtcgagga gggcctattt cccatgattc cttcatattt gcatatacga
      961 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
     1021 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     1081 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     1141 tatatcttgt ggaaaggacg aaacaccgat aacttcgtat agcatacatt atacgaagtt
     1201 atggtaccga tgcagctagc ccgcggatct gccggtctcc ctatagtgag tcgtattaat
     1261 ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1321 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     1381 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     1441 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1501 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1561 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1621 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1681 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     1741 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     1801 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     1861 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     1921 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     1981 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2041 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2101 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     2161 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     2221 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     2281 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     2341 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     2401 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     2461 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     2521 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     2581 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     2641 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     2701 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     2761 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     2821 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     2881 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     2941 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     3001 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     3061 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     3121 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     3181 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     3241 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     3301 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     3361 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     3421 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     3481 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga
     3541 acgcggctac aattaataca taaccttatg tatcatacac atacgattta ggtgacacta
     3601 ta
//
