LOCUS       sequence-228067-        5806 bp    DNA     circular SYN 15-APR-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 Apr 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"
     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..2270
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2121..2256
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2892..3116
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3123..3371
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3123..3143
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3294..3313
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(3373..3406)
                     /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(3443..3462)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3444..3462)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3549..3566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3720..4308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3800..3819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4479..5270,5271..5339))
                     /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     5102..5121
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5340..5444)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5512..5530
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5568..5590)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5690..5709
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5790..5806,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5790..5806,1)
                     /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 gacggtgtgg aaagtcccca ggctccccag
     1981 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2041 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2101 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     2161 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     2221 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gaattcgcca
     2281 ccatgactga gtacaagcct actgtgcgtt tagccaccag ggatgatgtg ccacgagccg
     2341 tgaggacact agcagccgcc tttgctgact acccagctac aaggcacacc gtggaccctg
     2401 acagacacat tgaaagagtc acagaacttc aggagctgtt tttaactagg gtgggcctgg
     2461 atattggcaa agtctgggtg gccgatgatg gagcagcagt tgcagtgtgg accaccccag
     2521 agtctgttga agctggagct gtctttgcag aaattggacc aagaatggcc gagttatctg
     2581 gcagcaggct ggctgctcaa cagcagatgg aaggattatt agctccccac agacccaagg
     2641 agcctgcctg gttcctggca acagttggag tttctccaga ccatcagggc aagggcttag
     2701 gatctgctgt ggtgctgcct ggtgtggaag cagctgagag agctggggtg ccagcatttc
     2761 tggagacatc agctcctaga aacctgccct tctatgaacg gctgggcttc acagtgactg
     2821 ctgatgtgga ggtgcctgag ggccccagaa cctggtgcat gacaagaaaa ccaggagcct
     2881 gactgcagcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2941 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     3001 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     3061 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctcg
     3121 aggagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     3181 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     3241 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     3301 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     3361 gacgaaacac cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc
     3421 tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt
     3481 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3541 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3601 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3661 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3721 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3781 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3841 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3901 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3961 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4021 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4081 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4141 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4201 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4261 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4321 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4381 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4441 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4501 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4561 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4621 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4681 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4741 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4801 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4861 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4921 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4981 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5041 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5101 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5161 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5221 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5281 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5341 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5401 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5461 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     5521 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     5581 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     5641 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     5701 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa
     5761 tacataacct tatgtatcat acacatacga tttaggtgac actata
//
