LOCUS       sequence-243814-        5536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mCherry-mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5536)
  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 5536)
  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-P19
FEATURES             Location/Qualifiers
     source          1..5536
                     /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..785
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(223..241)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     491..510
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             816..1526
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1071..1080
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1533..1654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1570..1589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1624..1643
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1661..1694)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1701..2017
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1868..2003
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2639..2863
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2870..3118
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2870..2890
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3041..3060
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(3120..3153)
                     /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(3190..3209)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3191..3209)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3296..3313)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3467..4055)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3547..3566)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4226..5017,5018..5086))
                     /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     4849..4868
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5087..5191)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5259..5277
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5315..5337)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5437..5456
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagga taacatggcc atcatcaagg
      121 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
      181 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
      241 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
      301 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
      361 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
      421 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
      481 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
      541 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
      601 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
      661 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca
      721 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt
      781 acaagggtgc aggagctgga accggtgcag gagctatggt gagcaagggc gaggaggata
      841 acatggcctc tctcccagcg acacatgagt tacacatctt tggctccatc aacggtgtgg
      901 actttgacat ggtgggtcag ggcaccggca atccaaatga tggttatgag gagttaaacc
      961 tgaagtccac caagggtgac ctccagttct ccccctggat tctggtccct catatcgggt
     1021 atggcttcca tcagtacctg ccctaccctg acgggatgtc gcctttccag gccgccatgg
     1081 tagatggctc cggataccaa gtccatcgca caatgcagtt tgaagatggt gcctccctta
     1141 ctgttaacta ccgctacacc tacgagggaa gccacatcaa aggagaggcc caggtgaagg
     1201 ggactggttt ccctgctgac ggtcctgtga tgaccaactc gctgaccgct gcggactggt
     1261 gcaggtcgaa gaagacttac cccaacgaca aaaccatcat cagtaccttt aagtggagtt
     1321 acaccactgg aaatggcaag cgctaccgga gcactgcgcg gaccacctac acctttgcca
     1381 agccaatggc ggctaactat ctgaagaacc agccgatgta cgtgttccgt aagacggagc
     1441 tcaagcactc caagaccgag ctcaacttca aggagtggca aaaggccttt accgatgtga
     1501 tgggcatgga cgagctgtac aagtgaacta gtaacttgtt tattgcagct tataatggtt
     1561 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     1621 gttgtggttt gtccaaactc atcaatgtat cttagtcgat ataacttcgt atagcataca
     1681 ttatacgaag ttatgtcgac ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     1741 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     1801 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     1861 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     1921 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     1981 gtgaggaggc ttttttggag gcctaggctt ttgcaaagaa ttcgccacca tgactgagta
     2041 caagcctact gtgcgtttag ccaccaggga tgatgtgcca cgagccgtga ggacactagc
     2101 agccgccttt gctgactacc cagctacaag gcacaccgtg gaccctgaca gacacattga
     2161 aagagtcaca gaacttcagg agctgttttt aactagggtg ggcctggata ttggcaaagt
     2221 ctgggtggcc gatgatggag cagcagttgc agtgtggacc accccagagt ctgttgaagc
     2281 tggagctgtc tttgcagaaa ttggaccaag aatggccgag ttatctggca gcaggctggc
     2341 tgctcaacag cagatggaag gattattagc tccccacaga cccaaggagc ctgcctggtt
     2401 cctggcaaca gttggagttt ctccagacca tcagggcaag ggcttaggat ctgctgtggt
     2461 gctgcctggt gtggaagcag ctgagagagc tggggtgcca gcatttctgg agacatcagc
     2521 tcctagaaac ctgcccttct atgaacggct gggcttcaca gtgactgctg atgtggaggt
     2581 gcctgagggc cccagaacct ggtgcatgac aagaaaacca ggagcctgac tgcagcgact
     2641 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2701 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2761 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2821 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggctcgagg agggcctatt
     2881 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     2941 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     3001 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     3061 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
     3121 taacttcgta tagcatacat tatacgaagt tatggtaccg atgcagctag cccgcggatc
     3181 tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat
     3241 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3301 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3361 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3421 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3481 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3541 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3601 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3661 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3721 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3781 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3841 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3901 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3961 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4021 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4081 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4141 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4201 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4261 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4321 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4381 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4441 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4501 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4561 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4621 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4681 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4741 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4801 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4861 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4921 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4981 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5041 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5101 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5161 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     5221 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     5281 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     5341 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     5401 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     5461 gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac ataaccttat
     5521 gtatcataca catacg
//
