LOCUS       sequence-232635-        4771 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mEos4b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4771)
  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 4771)
  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-P17
FEATURES             Location/Qualifiers
     source          1..4771
                     /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             81..761
                     /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    768..889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(805..824)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     859..878
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(896..929)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        936..1252
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1103..1238
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1874..2098
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2105..2353
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2105..2125
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2276..2295
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2355..2388)
                     /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(2425..2444)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2426..2444)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2531..2548)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2702..3290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2782..2801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3461..4252,4253..4321))
                     /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     4084..4103
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4322..4426)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4494..4512
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4550..4572)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4672..4691
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagtgcga ttaagccaga catgaggatc aaactccgta
      121 tggaaggcaa cgtaaacggg caccactttg tgatcgacgg agatggtaca ggcaagcctt
      181 atgagggaaa acagaccatg gatcttgaag tcaaagaggg cggacctctg ccttttgcct
      241 ttgatatcct gaccactgca ttccattacg gcaacagggt attcgtgaaa tatccagaca
      301 acatacaaga ctattttaag cagtcgtttc ctaaggggta ttcgtgggaa cgaagcttga
      361 ctttcgaaga cgggggcatt tgcaatgcca gaaacgacat aacaatggaa ggggacactt
      421 tctataataa agttcgattt tatggtacca actttcccgc caatggtcca gttatgcaga
      481 agaagacgct gaaatgggag ccctccactg agaaaatgta tgtgcgtgat ggagtgctga
      541 cgggtgatat tgagatggct ttgttgcttg aaggaaatgc ccattaccga tgtgacttca
      601 gaactactta caaagctaag gagaagggtg tcaagttacc aggcgcccac tttgtggacc
      661 acgccattga gattttaagc catgacaaag attacaacaa ggttaagctg tatgagcatg
      721 ctgttgctca ttctggattg cctgacaatg ccagacgatg aactagtaac ttgtttattg
      781 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
      841 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag tcgatataac
      901 ttcgtatagc atacattata cgaagttatg tcgacggtgt ggaaagtccc caggctcccc
      961 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     1021 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     1081 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     1141 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     1201 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagaattcgc
     1261 caccatgact gagtacaagc ctactgtgcg tttagccacc agggatgatg tgccacgagc
     1321 cgtgaggaca ctagcagccg cctttgctga ctacccagct acaaggcaca ccgtggaccc
     1381 tgacagacac attgaaagag tcacagaact tcaggagctg tttttaacta gggtgggcct
     1441 ggatattggc aaagtctggg tggccgatga tggagcagca gttgcagtgt ggaccacccc
     1501 agagtctgtt gaagctggag ctgtctttgc agaaattgga ccaagaatgg ccgagttatc
     1561 tggcagcagg ctggctgctc aacagcagat ggaaggatta ttagctcccc acagacccaa
     1621 ggagcctgcc tggttcctgg caacagttgg agtttctcca gaccatcagg gcaagggctt
     1681 aggatctgct gtggtgctgc ctggtgtgga agcagctgag agagctgggg tgccagcatt
     1741 tctggagaca tcagctccta gaaacctgcc cttctatgaa cggctgggct tcacagtgac
     1801 tgctgatgtg gaggtgcctg agggccccag aacctggtgc atgacaagaa aaccaggagc
     1861 ctgactgcag cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1921 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     1981 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2041 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2101 cgaggagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     2161 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     2221 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     2281 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     2341 aggacgaaac accgataact tcgtatagca tacattatac gaagttatgg taccgatgca
     2401 gctagcccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg
     2461 ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2521 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2581 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2641 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2701 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2761 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2821 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2881 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2941 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3001 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3061 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3121 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3181 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3241 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3301 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3361 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3421 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3481 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3541 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3601 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3661 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3721 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3781 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3841 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3901 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3961 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4021 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4081 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4141 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4201 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4261 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4321 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4381 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4441 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4501 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4561 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     4621 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     4681 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt
     4741 aatacataac cttatgtatc atacacatac g
//
