LOCUS       sequence-228135-        4546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mEos4b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4546)
  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 4546)
  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-Z17
FEATURES             Location/Qualifiers
     source          1..4546
                     /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..1235
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1086..1221
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1242..1251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1248..1622
                     /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    1632..1856
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1863..2111
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1863..1883
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2034..2053
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2113..2146)
                     /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(2183..2202)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2184..2202)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2289..2306)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2460..3048)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2540..2559)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3219..4010,4011..4079))
                     /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     3842..3861
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4080..4184)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4252..4270
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4308..4330)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4430..4449
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4530..4546,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4530..4546,1)
                     /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 atgtcgacgg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
      961 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     1021 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     1081 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     1141 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     1201 gaggaggctt ttttggaggc ctaggctttt gcaaagaatt cgccaccatg gccaagttga
     1261 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
     1321 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
     1381 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
     1441 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
     1501 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
     1561 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
     1621 gactgcagcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     1681 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     1741 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     1801 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctcg
     1861 aggagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     1921 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     1981 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     2041 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     2101 gacgaaacac cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc
     2161 tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt
     2221 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2281 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2341 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2401 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2461 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2521 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2581 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2641 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2701 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2761 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2821 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2881 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2941 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3001 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3061 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3121 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3181 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3241 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3301 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3361 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3421 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3481 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3541 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3601 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3661 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3721 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3781 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3841 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3901 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3961 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4021 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4081 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4141 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4201 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     4261 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     4321 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     4381 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     4441 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa
     4501 tacataacct tatgtatcat acacatacga tttaggtgac actata
//
