LOCUS       sequence-228063-        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"
     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"
     polyA_signal    1857..2081
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2088..2336
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2088..2108
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2259..2278
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2338..2371)
                     /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(2408..2427)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2409..2427)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2514..2531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2685..3273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2765..2784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3444..4235,4236..4304))
                     /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     4067..4086
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4305..4409)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4477..4495
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4533..4555)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4655..4674
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4755..4771,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4755..4771,1)
                     /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 actgagtaca
     1261 agcctactgt gcgtttagcc accagggatg atgtgccacg agccgtgagg acactagcag
     1321 ccgcctttgc tgactaccca gctacaaggc acaccgtgga ccctgacaga cacattgaaa
     1381 gagtcacaga acttcaggag ctgtttttaa ctagggtggg cctggatatt ggcaaagtct
     1441 gggtggccga tgatggagca gcagttgcag tgtggaccac cccagagtct gttgaagctg
     1501 gagctgtctt tgcagaaatt ggaccaagaa tggccgagtt atctggcagc aggctggctg
     1561 ctcaacagca gatggaagga ttattagctc cccacagacc caaggagcct gcctggttcc
     1621 tggcaacagt tggagtttct ccagaccatc agggcaaggg cttaggatct gctgtggtgc
     1681 tgcctggtgt ggaagcagct gagagagctg gggtgccagc atttctggag acatcagctc
     1741 ctagaaacct gcccttctat gaacggctgg gcttcacagt gactgctgat gtggaggtgc
     1801 ctgagggccc cagaacctgg tgcatgacaa gaaaaccagg agcctgactg cagcgactgt
     1861 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1921 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1981 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2041 agacaatagc aggcatgctg gggatgcggt gggctctatg gctcgaggag ggcctatttc
     2101 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     2161 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     2221 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     2281 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgata
     2341 acttcgtata gcatacatta tacgaagtta tggtaccgat gcagctagcc cgcggatctg
     2401 ccggtctccc tatagtgagt cgtattaatt tcgataagcc aggttaacct gcattaatga
     2461 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2521 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2581 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2641 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2701 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2761 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2821 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     2881 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2941 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3001 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3061 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3121 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3181 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3241 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3301 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3361 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3421 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3481 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3541 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3601 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3661 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3721 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3781 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3841 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3901 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3961 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4021 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4081 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4141 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4201 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4261 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4321 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4381 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4441 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     4501 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4561 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     4621 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     4681 gtgcaccata tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt
     4741 atcatacaca tacgatttag gtgacactat a
//
