LOCUS       sequence-228126-        4549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mClover3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4549)
  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 4549)
  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-Z08
FEATURES             Location/Qualifiers
     source          1..4549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(106..127)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     714..735
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    754..875
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(791..810)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     845..864
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(882..915)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        922..1238
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1089..1224
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1245..1254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1251..1625
                     /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    1635..1859
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1866..2114
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1866..1886
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2037..2056
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2116..2149)
                     /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(2186..2205)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2187..2205)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2292..2309)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2463..3051)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2543..2562)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3222..4013,4014..4082))
                     /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     3845..3864
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4083..4187)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4255..4273
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4311..4333)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4433..4452
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4533..4549,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4533..4549,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      121 ggcgacgtaa acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac
      181 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      241 ctcgtgacca ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag
      301 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct
      361 ttcaaggacg acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      421 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      481 aagctggagt acaacttcaa cagccactac gtctatatca cggccgacaa gcagaagaac
      541 tgcatcaagg ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc
      601 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      661 tacctgagcc atcagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      721 ctgctggagt tcgtgaccgc cgcctgaact agtaacttgt ttattgcagc ttataatggt
      781 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
      841 agttgtggtt tgtccaaact catcaatgta tcttagtcga tataacttcg tatagcatac
      901 attatacgaa gttatgtcga cggtgtggaa agtccccagg ctccccagca ggcagaagta
      961 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     1021 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     1081 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     1141 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     1201 agtgaggagg cttttttgga ggcctaggct tttgcaaaga attcgccacc atggccaagt
     1261 tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga
     1321 ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg
     1381 acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg
     1441 cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca
     1501 cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc
     1561 gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg
     1621 actgactgca gcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     1681 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     1741 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     1801 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     1861 tcgaggaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     1921 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     1981 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     2041 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     2101 aaggacgaaa caccgataac ttcgtatagc atacattata cgaagttatg gtaccgatgc
     2161 agctagcccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag
     2221 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2281 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2341 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2401 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2461 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2521 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2581 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2641 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2701 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2761 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2821 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2881 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     2941 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3001 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3061 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3121 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3181 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3241 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3301 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3361 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3421 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3481 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3541 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3601 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3661 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3721 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3781 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3841 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3901 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3961 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4021 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4081 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4141 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4201 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     4261 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     4321 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     4381 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     4441 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat
     4501 taatacataa ccttatgtat catacacata cgatttaggt gacactata
//
