LOCUS       sequence-232705-        5581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with 24xSunTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5581)
  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 5581)
  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-Z21
FEATURES             Location/Qualifiers
     source          1..5581
                     /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..137
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             153..209
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             225..281
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             297..353
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             369..425
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             441..497
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             513..569
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             585..641
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             657..713
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             729..785
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             801..857
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             873..929
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             945..1001
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1017..1073
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1089..1145
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1161..1217
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1233..1289
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1305..1361
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1377..1433
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1449..1505
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1521..1577
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1593..1649
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1737..1793
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     polyA_signal    1803..1924
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1840..1859)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1894..1913
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1931..1964)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1971..2287
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2138..2273
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      2294..2303
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2300..2674
                     /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    2684..2908
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2915..3163
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2915..2935
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3086..3105
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(3165..3198)
                     /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(3235..3254)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3236..3254)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3341..3358)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3512..4100)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3592..3611)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4271..5062,5063..5131))
                     /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     4894..4913
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5132..5236)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5304..5322
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5360..5382)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5482..5501
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gaagaacttt tgagcaagaa ttatcatctt gagaacgaag
      121 tggctcgtct taagaaaggt tctggcagtg gagaagaact gctttcaaag aattaccacc
      181 tggaaaatga ggtagctaga ctgaaaaagg ggagcggaag tggggaggag ttgctgagca
      241 aaaattatca tttggagaac gaagtagcac gactaaagaa agggtccgga tcgggtgagg
      301 agttactctc gaaaaattat catctcgaaa acgaagtggc tcggctaaaa aagggcagtg
      361 gttctggaga agagctatta tctaaaaact accacctcga aaatgaggtg gcacgcttaa
      421 aaaagggaag tggcagtggt gaagagctac tatccaagaa ttatcatctt gagaacgagg
      481 tagcgcgttt gaagaagggt tccggctcag gagaggaact gctctcgaag aactatcatc
      541 ttgaaaatga ggtcgctcga ttaaaaaagg gatcgggcag tggtgaggaa ctactttcaa
      601 agaattacca cctcgaaaac gaagtagctc gattaaagaa aggttcaggg tcgggtgaag
      661 aattactgag taaaaattat catctggaaa atgaggtagc gagactaaaa aaggggagtg
      721 gttctggcga agagttgcta tcgaaaaatt atcatcttga gaacgaagtt gctaggctca
      781 aaaagggctc aggctcaggc gaggagttgc tctcgaaaaa ctaccacttg gaaaatgagg
      841 tcgcgaggtt gaaaaagggg agcgggtcgg gcgaggagtt attgagcaaa aactatcatt
      901 tagagaacga agtcgcgcgc ttaaagaaag gctcgggctc gggcgaagaa ctcttatcga
      961 agaactacca cctcgaaaat gaggtcgcca ggttgaaaaa gggcagtggc agcggggagg
     1021 aactcttgag caagaactac cacttggaga atgaggtcgc gagattgaag aaagggtcgg
     1081 ggagcggcga ggaattgctc agcaagaatt atcatttgga gaacgaagtc gccaggctca
     1141 agaaaggctc ggggtcgggg gaggaattgt tgagtaaaaa ctaccacttg gaaaatgaag
     1201 tcgccaggct caaaaaaggg agtgggagcg gcgaagagtt attgagcaaa aattaccact
     1261 tggagaacga agtggcaagg ctcaagaaag ggagcggcag cggggaggag ctcttatcga
     1321 agaactacca cttagagaat gaagtcgccc gcttgaagaa aggctcgggg agcggggaag
     1381 agctcttgag caagaactac cacttggaaa atgaggtggc gcgcttgaag aaagggagcg
     1441 ggagcgggga agagttacta tctaagaatt atcatctcga gaacgaggtg gctcgactaa
     1501 agaagggctc cggcagtggg gaggaactcc tgtcgaagaa ctatcatctt gaaaatgagg
     1561 ttgcaagact taaaaagggg tccggatcag gtgaggaact actcagtaag aattaccacc
     1621 tggaaaacga agttgcacgt ttgaagaaag gatcaggatc aggcgaagaa ctgctctcaa
     1681 aagattatca tttggaaaat gaggttgcac gtttaaaaaa gggaagtggc agtggtgagg
     1741 aacttctgtc gaaaaattat catctcgaga atgaagtagc ccgacttaaa aagtgaacta
     1801 gtaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     1861 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     1921 cttagtcgat ataacttcgt atagcataca ttatacgaag ttatgtcgac ggtgtggaaa
     1981 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2041 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2101 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     2161 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     2221 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     2281 ttgcaaagaa ttcgccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     2341 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     2401 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     2461 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     2521 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     2581 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     2641 ctgcgtgcac ttcgtggccg aggagcagga ctgactgcag cgactgtgcc ttctagttgc
     2701 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2761 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2821 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2881 catgctgggg atgcggtggg ctctatggct cgaggagggc ctatttccca tgattccttc
     2941 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     3001 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     3061 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     3121 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgataact tcgtatagca
     3181 tacattatac gaagttatgg taccgatgca gctagcccgc ggatctgccg gtctccctat
     3241 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg
     3301 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3361 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3421 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3481 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3541 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3601 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3661 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3721 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3781 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3841 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3901 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3961 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4021 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4081 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4141 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4201 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4261 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4321 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4381 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4441 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4501 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4561 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4621 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4681 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4741 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4801 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4861 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4921 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4981 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5041 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5101 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5161 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5221 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     5281 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     5341 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     5401 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     5461 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg
     5521 acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac
     5581 g
//
