LOCUS       sequence-228026-        4637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with 24xSunTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4637)
  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 4637)
  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-21
FEATURES             Location/Qualifiers
     source          1..4637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..120
                     /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             136..192
                     /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             208..264
                     /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             280..336
                     /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             352..408
                     /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             424..480
                     /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             496..552
                     /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             568..624
                     /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             640..696
                     /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             712..768
                     /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             784..840
                     /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             856..912
                     /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             928..984
                     /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             1000..1056
                     /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             1072..1128
                     /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             1144..1200
                     /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             1216..1272
                     /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             1288..1344
                     /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             1360..1416
                     /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             1432..1488
                     /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             1504..1560
                     /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             1576..1632
                     /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             1720..1776
                     /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    1786..1907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1823..1842)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1877..1896
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1914..1947)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1954..2202
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1954..1974
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2125..2144
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2204..2237)
                     /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(2274..2293)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2275..2293)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2380..2397)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2551..3139)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2631..2650)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3310..4101,4102..4170))
                     /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     3933..3952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4171..4275)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4343..4361
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4399..4421)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4521..4540
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4621..4637,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4621..4637,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgaagaac ttttgagcaa gaattatcat cttgagaacg aagtggctcg tcttaagaaa
      121 ggttctggca gtggagaaga actgctttca aagaattacc acctggaaaa tgaggtagct
      181 agactgaaaa aggggagcgg aagtggggag gagttgctga gcaaaaatta tcatttggag
      241 aacgaagtag cacgactaaa gaaagggtcc ggatcgggtg aggagttact ctcgaaaaat
      301 tatcatctcg aaaacgaagt ggctcggcta aaaaagggca gtggttctgg agaagagcta
      361 ttatctaaaa actaccacct cgaaaatgag gtggcacgct taaaaaaggg aagtggcagt
      421 ggtgaagagc tactatccaa gaattatcat cttgagaacg aggtagcgcg tttgaagaag
      481 ggttccggct caggagagga actgctctcg aagaactatc atcttgaaaa tgaggtcgct
      541 cgattaaaaa agggatcggg cagtggtgag gaactacttt caaagaatta ccacctcgaa
      601 aacgaagtag ctcgattaaa gaaaggttca gggtcgggtg aagaattact gagtaaaaat
      661 tatcatctgg aaaatgaggt agcgagacta aaaaagggga gtggttctgg cgaagagttg
      721 ctatcgaaaa attatcatct tgagaacgaa gttgctaggc tcaaaaaggg ctcaggctca
      781 ggcgaggagt tgctctcgaa aaactaccac ttggaaaatg aggtcgcgag gttgaaaaag
      841 gggagcgggt cgggcgagga gttattgagc aaaaactatc atttagagaa cgaagtcgcg
      901 cgcttaaaga aaggctcggg ctcgggcgaa gaactcttat cgaagaacta ccacctcgaa
      961 aatgaggtcg ccaggttgaa aaagggcagt ggcagcgggg aggaactctt gagcaagaac
     1021 taccacttgg agaatgaggt cgcgagattg aagaaagggt cggggagcgg cgaggaattg
     1081 ctcagcaaga attatcattt ggagaacgaa gtcgccaggc tcaagaaagg ctcggggtcg
     1141 ggggaggaat tgttgagtaa aaactaccac ttggaaaatg aagtcgccag gctcaaaaaa
     1201 gggagtggga gcggcgaaga gttattgagc aaaaattacc acttggagaa cgaagtggca
     1261 aggctcaaga aagggagcgg cagcggggag gagctcttat cgaagaacta ccacttagag
     1321 aatgaagtcg cccgcttgaa gaaaggctcg gggagcgggg aagagctctt gagcaagaac
     1381 taccacttgg aaaatgaggt ggcgcgcttg aagaaaggga gcgggagcgg ggaagagtta
     1441 ctatctaaga attatcatct cgagaacgag gtggctcgac taaagaaggg ctccggcagt
     1501 ggggaggaac tcctgtcgaa gaactatcat cttgaaaatg aggttgcaag acttaaaaag
     1561 gggtccggat caggtgagga actactcagt aagaattacc acctggaaaa cgaagttgca
     1621 cgtttgaaga aaggatcagg atcaggcgaa gaactgctct caaaagatta tcatttggaa
     1681 aatgaggttg cacgtttaaa aaagggaagt ggcagtggtg aggaacttct gtcgaaaaat
     1741 tatcatctcg agaatgaagt agcccgactt aaaaagtgaa ctagtaactt gtttattgca
     1801 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     1861 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttagtc gatataactt
     1921 cgtatagcat acattatacg aagttatgtc gaggagggcc tatttcccat gattccttca
     1981 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
     2041 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     2101 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     2161 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgataactt cgtatagcat
     2221 acattatacg aagttatggt accgatgcag ctagcccgcg gatctgccgg tctccctata
     2281 gtgagtcgta ttaatttcga taagccaggt taacctgcat taatgaatcg gccaacgcgc
     2341 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     2401 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2461 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     2521 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2581 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2641 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2701 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     2761 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2821 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2881 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2941 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     3001 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3061 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3121 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3181 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3241 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3301 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3361 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3421 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3481 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3541 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3601 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3661 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3721 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3781 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3841 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3901 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3961 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4021 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4081 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4141 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4201 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4261 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     4321 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     4381 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     4441 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     4501 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgga
     4561 catattgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg
     4621 atttaggtga cactata
//
