LOCUS       sequence-337972-        3998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal donor plasmid for CRISPitope method encoding mNeon 
            fluorescent protein, FLAG tag, human gp100 CD8+ T cell epitope [AA: 
            25-33 (KVPRNQDWL)] and puromycin resistance cassette [p.M1G].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3998)
  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 3998)
  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-hgp100-PuroR [M1G]
FEATURES             Location/Qualifiers
     source          1..3998
                     /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             1028..1081
                     /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"
     CDS             1085..1681
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1578..1598
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1706..1827
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1717..1736)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1771..1790
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2075..2092)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2246..2834)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2326..2345)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3034..3825,3826..3894))
                     /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 tgaaagtgcc gcgcaaccag gattggctgg tgtacaaggg
     1021 atccggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc
     1081 agggaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     1141 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     1201 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     1261 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     1321 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     1381 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     1441 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     1501 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     1561 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     1621 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     1681 agcggcccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     1741 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     1801 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     1861 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa
     1921 atcgataagg atccgtcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     1981 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     2041 cgtaggacag gtgccggcag cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2101 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2161 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2221 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2281 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2341 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2401 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2461 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2521 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2581 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2641 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2701 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2761 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2821 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2881 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2941 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3001 acagcggccg caaatgctaa accactgcag tggttaccag tgcttgatca gtgaggcacc
     3061 gatctcagcg atctgcctat ttcgttcgtc catagtggcc tgactccccg tcgtgtagat
     3121 cactacgatt cgtgagggct taccatcagg ccccagcgca gcaatgatgc cgcgagagcc
     3181 gcgttcaccg gcccccgatt tgtcagcaat gaaccagcca gcagggaggg ccgagcgaag
     3241 aagtggtcct gctactttgt ccgcctccat ccagtctatg agctgctgtc gtgatgctag
     3301 agtaagaagt tcgccagtga gtagtttccg aagagttgtg gccattgcta ctggcatcgt
     3361 ggtatcacgc tcgtcgttcg gtatggcttc gttcaactct ggttcccagc ggtcaagccg
     3421 ggtcacatga tcacccatat tatgaagaaa tgcagtcagc tccttagggc ctccgatcgt
     3481 tgtcagaagt aagttggccg cggtgttgtc gctcatggta atggcagcac tacacaattc
     3541 tcttaccgtc atgccatccg taagatgctt ttccgtgacc ggcgagtact caaccaagtc
     3601 gttttgtgag tagtgtatac ggcgaccaag ctgctcttgc ccggcgtcta tacgggacaa
     3661 caccgcgcca catagcagta ctttgaaagt gctcatcatc gggaatcgtt cttcggggcg
     3721 gaaagactca aggatcttgc cgctattgag atccagttcg atatagccca ctcttgcacc
     3781 cagttgatct tcagcatctt ttactttcac cagcgtttcg gggtgtgcaa aaacaggcaa
     3841 gcaaaatgcc gcaaagaagg gaatgagtgc gacacgaaaa tgttggatgc tcatactcgt
     3901 cctttttcaa tattattgaa gcatttatca gagttactag tacgtctctc aaggataagt
     3961 aagtaatatt aaggtacggg aggtattgga caggccgc
//
