LOCUS       sequence-232699-        5806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with 24xSunTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5806)
  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 5806)
  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-P21
FEATURES             Location/Qualifiers
     source          1..5806
                     /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"
     polyA_signal    2909..3133
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3140..3388
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3140..3160
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3311..3330
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(3390..3423)
                     /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(3460..3479)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3461..3479)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3566..3583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3737..4325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3817..3836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4496..5287,5288..5356))
                     /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     5119..5138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5357..5461)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5529..5547
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5585..5607)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5707..5726
                     /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 tgactgagta caagcctact gtgcgtttag ccaccaggga
     2341 tgatgtgcca cgagccgtga ggacactagc agccgccttt gctgactacc cagctacaag
     2401 gcacaccgtg gaccctgaca gacacattga aagagtcaca gaacttcagg agctgttttt
     2461 aactagggtg ggcctggata ttggcaaagt ctgggtggcc gatgatggag cagcagttgc
     2521 agtgtggacc accccagagt ctgttgaagc tggagctgtc tttgcagaaa ttggaccaag
     2581 aatggccgag ttatctggca gcaggctggc tgctcaacag cagatggaag gattattagc
     2641 tccccacaga cccaaggagc ctgcctggtt cctggcaaca gttggagttt ctccagacca
     2701 tcagggcaag ggcttaggat ctgctgtggt gctgcctggt gtggaagcag ctgagagagc
     2761 tggggtgcca gcatttctgg agacatcagc tcctagaaac ctgcccttct atgaacggct
     2821 gggcttcaca gtgactgctg atgtggaggt gcctgagggc cccagaacct ggtgcatgac
     2881 aagaaaacca ggagcctgac tgcagcgact gtgccttcta gttgccagcc atctgttgtt
     2941 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3001 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3061 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     3121 gtgggctcta tggctcgagg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     3181 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     3241 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     3301 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     3361 atatatcttg tggaaaggac gaaacaccga taacttcgta tagcatacat tatacgaagt
     3421 tatggtaccg atgcagctag cccgcggatc tgccggtctc cctatagtga gtcgtattaa
     3481 tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3541 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3601 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3661 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3721 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3781 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3841 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3901 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3961 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4021 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4081 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4141 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4201 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4261 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4321 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4381 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4441 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4501 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4561 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4621 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     4681 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     4741 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4801 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4861 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4921 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4981 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5041 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5101 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5161 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5221 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5281 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5341 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5401 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5461 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     5521 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     5581 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     5641 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     5701 actatgcggc atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag
     5761 aacgcggcta caattaatac ataaccttat gtatcataca catacg
//
