LOCUS       sequence-243822-        5311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mCherry-mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5311)
  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 5311)
  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-Z19
FEATURES             Location/Qualifiers
     source          1..5311
                     /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"
     regulatory      2024..2033
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2030..2404
                     /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    2414..2638
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2645..2893
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2645..2665
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2816..2835
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2895..2928)
                     /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(2965..2984)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2966..2984)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3071..3088)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3242..3830)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3322..3341)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4001..4792,4793..4861))
                     /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     4624..4643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4862..4966)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5034..5052
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5090..5112)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5212..5231
                     /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 tggccaagtt
     2041 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac
     2101 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga
     2161 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc
     2221 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac
     2281 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg
     2341 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga
     2401 ctgactgcag cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2461 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2521 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2581 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2641 cgaggagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     2701 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     2761 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     2821 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     2881 aggacgaaac accgataact tcgtatagca tacattatac gaagttatgg taccgatgca
     2941 gctagcccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg
     3001 ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3061 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3121 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3181 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3241 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3301 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3361 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3421 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3481 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3541 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3601 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3661 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3721 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3781 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3841 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3901 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3961 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4021 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4081 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4141 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4201 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4261 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4321 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4381 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4441 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4501 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4561 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4621 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4681 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4741 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4801 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4861 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4921 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4981 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5041 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     5101 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     5161 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     5221 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt
     5281 aatacataac cttatgtatc atacacatac g
//
