LOCUS       sequence-337979-        3852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal donor plasmid for CRISPitope method encoding mNeon 
            fluorescent protein, FLAG tag, HSV-1 glycoprotein B CD8+ T cell 
            epitope [AA: 498-505 (SSIEFARL)] and blasticidin resistance cassette
            [p.M1G.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3852)
  AUTHORS   Effern M, Glodde N, Braun M, Liebing J, Boll HN, Yong M, Bawden E, 
            Hinze D, van den Boorn-Konijnenberg D, Daoud M, Aymans P, Landsberg 
            J, Smyth MJ, Flatz L, Tuting T, Bald T, Gebhardt T, Holzel M
  TITLE     Adoptive T Cell Therapy Targeting Different Gene Products Reveals 
            Diverse and Context-Dependent Immune Evasion in Melanoma.
  JOURNAL   Immunity. 2020 Sep 15;53(3):564-580.e9. doi: 
            10.1016/j.immuni.2020.07.007. Epub 2020 Aug 3.
  PUBMED    32750334
REFERENCE   2  (bases 1 to 3852)
  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"; doi: 
            "10.1016/j.immuni.2020.07.007"; journalName: "Immunity"; date: 
            "2020-09-15- 15"; volume: "53"; issue: "3"; pages: "564-580.e9"
COMMENT     Sequence Label: pCRISPaint-mNeon-F-gB-BlastR [M1G]
FEATURES             Location/Qualifiers
     source          1..3852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..49
                     /note="synthetic polyadenylation signal"
     misc_feature    63..154
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     103..122
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     CDS             227..928
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEEL
                     NLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
                     SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTF
                     KWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKA
                     FTDVMGMDELYK"
     regulatory      476..485
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             941..964
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1025..1078
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     polyA_signal    1560..1681
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1571..1590)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1625..1644
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1929..1946)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2100..2688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2180..2199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2888..3679,3680..3748))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta
       61 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc
      121 cccagtgcaa gtgcaggtgc cagaacattt ctcttaatga tagctaggat cgctagcggg
      181 ccagtaccca aaaagcgggg ggtctggtgg cagtggaggg ggatccgtga gcaagggcga
      241 ggaggataac gcctctctcc cagcgacaca tgagttacac atctttggct ccatcaacgg
      301 tgtggacttt gacatggtgg gtcagggcac cggcaatcca aatgatggtt atgaggagtt
      361 aaacctgaag tccaccaagg gtgacctcca gttctccccc tggattctgg tccctcatat
      421 cgggtatggc ttccatcagt acctgcccta ccctgacggg atgtcgcctt tccaggccgc
      481 catggtagat ggctccggat accaagtcca tcgcacaatg cagtttgaag atggtgcctc
      541 ccttactgtt aactaccgct acacctacga gggaagccac atcaaaggag aggcccaggt
      601 gaaggggact ggtttccctg ctgacggtcc tgtgatgacc aactcgctga ccgctgcgga
      661 ctggtgcagg tcgaagaaga cttaccccaa cgacaaaacc atcatcagta cctttaagtg
      721 gagttacacc actggaaatg gcaagcgcta ccggagcact gcgcggacca cctacacctt
      781 tgccaagcca atggcggcta actatctgaa gaaccagccg atgtacgtgt tccgtaagac
      841 ggagctcaag cactccaaga ccgagctcaa cttcaaggag tggcaaaagg cctttaccga
      901 tgtgatgggc atggacgagc tgtacaagag cggctccgcc gattacaagg atgacgacga
      961 caaacgatcg gggcctaggc tgagcagcat cgagttcgcc agactggtgt acaagggatc
     1021 cggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccagg
     1081 gaagaccttc aacatctctc agcaggatct ggagctggtg gaggtcgcca ctgagaagat
     1141 caccatgctc tatgaggaca acaagcacca tgtcggggcg gccatcagga ccaagactgg
     1201 ggagatcatc tctgctgtcc acattgaggc ctacattggc agggtcactg tctgtgctga
     1261 agccattgcc attgggtctg ctgtgagcaa cgggcagaag gactttgaca ccattgtggc
     1321 tgtcaggcac ccctactctg atgaggtgga cagatccatc agggtggtca gcccctgtgg
     1381 catgtgcaga gagctcatct ctgactatgc tcctgactgc tttgtgctca ttgagatgaa
     1441 tggcaagctg gtcaaaacca ccattgagga actcatcccc ctcaagtaca ccaggaacta
     1501 acacctggtg catgacccgc aagcccggtg cctgagcggc ccgcttcgag cagacatgat
     1561 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     1621 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     1681 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     1741 ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatccgt cgaccgatgc
     1801 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
     1861 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     1921 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1981 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2041 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2101 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2161 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2221 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2281 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2341 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2401 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2461 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2521 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2581 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2641 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2701 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2761 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2821 tcaatctaaa gtatatatga gtaaacttgg tctgacagcg gccgcaaatg ctaaaccact
     2881 gcagtggtta ccagtgcttg atcagtgagg caccgatctc agcgatctgc ctatttcgtt
     2941 cgtccatagt ggcctgactc cccgtcgtgt agatcactac gattcgtgag ggcttaccat
     3001 caggccccag cgcagcaatg atgccgcgag agccgcgttc accggccccc gatttgtcag
     3061 caatgaacca gccagcaggg agggccgagc gaagaagtgg tcctgctact ttgtccgcct
     3121 ccatccagtc tatgagctgc tgtcgtgatg ctagagtaag aagttcgcca gtgagtagtt
     3181 tccgaagagt tgtggccatt gctactggca tcgtggtatc acgctcgtcg ttcggtatgg
     3241 cttcgttcaa ctctggttcc cagcggtcaa gccgggtcac atgatcaccc atattatgaa
     3301 gaaatgcagt cagctcctta gggcctccga tcgttgtcag aagtaagttg gccgcggtgt
     3361 tgtcgctcat ggtaatggca gcactacaca attctcttac cgtcatgcca tccgtaagat
     3421 gcttttccgt gaccggcgag tactcaacca agtcgttttg tgagtagtgt atacggcgac
     3481 caagctgctc ttgcccggcg tctatacggg acaacaccgc gccacatagc agtactttga
     3541 aagtgctcat catcgggaat cgttcttcgg ggcggaaaga ctcaaggatc ttgccgctat
     3601 tgagatccag ttcgatatag cccactcttg cacccagttg atcttcagca tcttttactt
     3661 tcaccagcgt ttcggggtgt gcaaaaacag gcaagcaaaa tgccgcaaag aagggaatga
     3721 gtgcgacacg aaaatgttgg atgctcatac tcgtcctttt tcaatattat tgaagcattt
     3781 atcagagtta ctagtacgtc tctcaaggat aagtaagtaa tattaaggta cgggaggtat
     3841 tggacaggcc gc
//
