LOCUS       sequence-232670-        4531 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mKeima8.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4531)
  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 4531)
  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-Z18
FEATURES             Location/Qualifiers
     source          1..4531
                     /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             join(81..83,84..743)
                     /codon_start=1
                     /product="human codon-optimized monomeric red fluorescent 
                     protein with a large Stokes shift (Guan et al. 2015)"
                     /label=hmKeima8.5
                     /translation="VSVIAEHMTYKVYMSGTVNGHYFEVEGDGEGKPYEGEQTVKLTVT
                     KGGPLPFAWHILSPQLQYGSIPFTKYPADIPDYFKQSFPEGYTWVRSMNFEDGAVCTVS
                     NDSSIRGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERMFARDGMLIGNDHMALKL
                     EGGGHYLCEFKSAYKARKPVRMPGLYEIDRKLDVTSHNRDYTSVEQREIAIARHPLLG"
     polyA_signal    753..874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(790..809)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     844..863
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(881..914)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        921..1237
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1088..1223
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1250..1624
                     /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    1634..1858
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1865..2113
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1865..1885
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2036..2055
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2115..2148)
                     /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(2185..2204)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2186..2204)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2291..2308)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2462..3050)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2542..2561)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3221..4012,4013..4081))
                     /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     3844..3863
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4082..4186)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4254..4272
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4310..4332)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4432..4451
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcgtga tcgccgagca catgacctac aaggtgtaca
      121 tgagcggcac cgtgaacggc cactacttcg aggtggaggg cgacggcgag ggcaagccct
      181 acgagggcga gcagaccgtg aagctgaccg tgaccaaggg tggccccctg cccttcgcct
      241 ggcacatcct gagcccccag ctccagtacg gcagcatccc cttcaccaag taccccgccg
      301 acatccccga ctacttcaag cagagcttcc ccgagggcta cacctgggtg cgcagcatga
      361 acttcgagga cggcgccgtg tgcaccgtga gcaacgacag cagcatccgc ggcaactgct
      421 tcatctacaa cgtgaagatc agcggcgaga acttcccccc caacggcccc gtgatgcaga
      481 agaagaccca gggctgggag cccagcaccg agcgcatgtt cgcccgcgac ggaatgctga
      541 tcggcaacga ccacatggcc ctgaagctgg agggcggcgg ccactacctg tgcgagttca
      601 agagcgccta caaggcccgc aagcccgtga ggatgcccgg cctgtacgag atcgaccgca
      661 agctggacgt gaccagccac aaccgcgact acaccagcgt ggagcagcgc gagatcgcca
      721 tcgcccgcca ccccctgctg ggctgaacta gtaacttgtt tattgcagct tataatggtt
      781 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
      841 gttgtggttt gtccaaactc atcaatgtat cttagtcgat ataacttcgt atagcataca
      901 ttatacgaag ttatgtcgac ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
      961 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     1021 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     1081 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     1141 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     1201 gtgaggaggc ttttttggag gcctaggctt ttgcaaagaa ttcgccacca tggccaagtt
     1261 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac
     1321 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga
     1381 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc
     1441 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac
     1501 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg
     1561 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga
     1621 ctgactgcag cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1681 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     1741 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     1801 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     1861 cgaggagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     1921 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     1981 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     2041 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     2101 aggacgaaac accgataact tcgtatagca tacattatac gaagttatgg taccgatgca
     2161 gctagcccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg
     2221 ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2281 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2341 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2401 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2461 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2521 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2581 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2641 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2701 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2761 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2821 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2881 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2941 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3001 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3061 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3121 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3181 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3241 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3301 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3361 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3421 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3481 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3541 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3601 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3661 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3721 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3781 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3841 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3901 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3961 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4021 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4081 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4141 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4201 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4261 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4321 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     4381 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     4441 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt
     4501 aatacataac cttatgtatc atacacatac g
//
