LOCUS       sequence-341955-        3995 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 puromycin resistance cassette 
            [p.M1G].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3995)
  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 3995)
  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-PuroR [M1G]
FEATURES             Location/Qualifiers
     source          1..3995
                     /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"
     CDS             1082..1678
                     /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     1575..1595
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1703..1824
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1714..1733)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1768..1787
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2072..2089)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2243..2831)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2323..2342)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3031..3822,3823..3891))
                     /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 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     1141 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     1201 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     1261 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     1321 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     1381 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     1441 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     1501 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     1561 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     1621 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgagc
     1681 ggcccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     1741 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     1801 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     1861 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     1921 gataaggatc cgtcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg
     1981 ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt
     2041 aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     2101 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2161 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2221 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2281 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2341 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     2401 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     2461 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2521 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2581 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2641 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     2701 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     2761 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     2821 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     2881 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     2941 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3001 gcggccgcaa atgctaaacc actgcagtgg ttaccagtgc ttgatcagtg aggcaccgat
     3061 ctcagcgatc tgcctatttc gttcgtccat agtggcctga ctccccgtcg tgtagatcac
     3121 tacgattcgt gagggcttac catcaggccc cagcgcagca atgatgccgc gagagccgcg
     3181 ttcaccggcc cccgatttgt cagcaatgaa ccagccagca gggagggccg agcgaagaag
     3241 tggtcctgct actttgtccg cctccatcca gtctatgagc tgctgtcgtg atgctagagt
     3301 aagaagttcg ccagtgagta gtttccgaag agttgtggcc attgctactg gcatcgtggt
     3361 atcacgctcg tcgttcggta tggcttcgtt caactctggt tcccagcggt caagccgggt
     3421 cacatgatca cccatattat gaagaaatgc agtcagctcc ttagggcctc cgatcgttgt
     3481 cagaagtaag ttggccgcgg tgttgtcgct catggtaatg gcagcactac acaattctct
     3541 taccgtcatg ccatccgtaa gatgcttttc cgtgaccggc gagtactcaa ccaagtcgtt
     3601 ttgtgagtag tgtatacggc gaccaagctg ctcttgcccg gcgtctatac gggacaacac
     3661 cgcgccacat agcagtactt tgaaagtgct catcatcggg aatcgttctt cggggcggaa
     3721 agactcaagg atcttgccgc tattgagatc cagttcgata tagcccactc ttgcacccag
     3781 ttgatcttca gcatctttta ctttcaccag cgtttcgggg tgtgcaaaaa caggcaagca
     3841 aaatgccgca aagaagggaa tgagtgcgac acgaaaatgt tggatgctca tactcgtcct
     3901 ttttcaatat tattgaagca tttatcagag ttactagtac gtctctcaag gataagtaag
     3961 taatattaag gtacgggagg tattggacag gccgc
//
