LOCUS       sequence-232595-        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"
     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..1184
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     936..956
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1107..1126
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1186..1219)
                     /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(1256..1275)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1257..1275)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1362..1379)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1533..2121)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1613..1632)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2292..3083,3084..3152))
                     /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     2915..2934
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3153..3257)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3325..3343
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3381..3403)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3503..3522
                     /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 tcgaggaggg cctatttccc atgattcctt
      961 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     1021 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     1081 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     1141 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgataac ttcgtatagc
     1201 atacattata cgaagttatg gtaccgatgc agctagcccg cggatctgcc ggtctcccta
     1261 tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc
     1321 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     1381 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     1441 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     1501 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     1561 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     1621 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     1681 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     1741 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     1801 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     1861 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     1921 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     1981 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     2041 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     2101 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     2161 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     2221 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     2281 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     2341 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2401 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     2461 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     2521 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     2581 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     2641 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     2701 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     2761 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     2821 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     2881 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     2941 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3001 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3061 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     3121 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     3181 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     3241 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     3301 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     3361 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     3421 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     3481 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     3541 gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat catacacata
     3601 cg
//
