LOCUS       sequence-228139-        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"
     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"
     regulatory      2277..2286
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2283..2657
                     /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    2667..2891
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2898..3146
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2898..2918
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3069..3088
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(3148..3181)
                     /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(3218..3237)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3219..3237)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3324..3341)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3495..4083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3575..3594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4254..5045,5046..5114))
                     /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     4877..4896
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5115..5219)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5287..5305
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5343..5365)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5465..5484
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5565..5581,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5565..5581,1)
                     /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 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
     2341 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
     2401 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
     2461 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
     2521 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
     2581 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
     2641 ccgaggagca ggactgactg cagcgactgt gccttctagt tgccagccat ctgttgtttg
     2701 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2761 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     2821 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     2881 gggctctatg gctcgaggag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2941 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     3001 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     3061 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     3121 atatcttgtg gaaaggacga aacaccgata acttcgtata gcatacatta tacgaagtta
     3181 tggtaccgat gcagctagcc cgcggatctg ccggtctccc tatagtgagt cgtattaatt
     3241 tcgataagcc aggttaacct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3301 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3361 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3421 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3481 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3541 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3601 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3661 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     3721 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3781 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3841 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3901 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     3961 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4021 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4081 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4141 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4201 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4261 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4321 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4381 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4441 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4501 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4561 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4621 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4681 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4741 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4801 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4861 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4921 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4981 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5041 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5101 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5161 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5221 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     5281 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     5341 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     5401 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     5461 tatgcggcat cagagcagat tgtactgaga gtgcaccata tggacatatt gtcgttagaa
     5521 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
     5581 a
//
