LOCUS       sequence-337977-        3855 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 blasticidin resistance cassette [p.M1G].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3855)
  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 3855)
  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-BlastR [M1G]
FEATURES             Location/Qualifiers
     source          1..3855
                     /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"
     polyA_signal    1563..1684
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1574..1593)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1628..1647
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1932..1949)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2103..2691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2183..2202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2891..3682,3683..3751))
                     /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 agggaagacc ttcaacatct ctcagcagga tctggagctg gtggaggtcg ccactgagaa
     1141 gatcaccatg ctctatgagg acaacaagca ccatgtcggg gcggccatca ggaccaagac
     1201 tggggagatc atctctgctg tccacattga ggcctacatt ggcagggtca ctgtctgtgc
     1261 tgaagccatt gccattgggt ctgctgtgag caacgggcag aaggactttg acaccattgt
     1321 ggctgtcagg cacccctact ctgatgaggt ggacagatcc atcagggtgg tcagcccctg
     1381 tggcatgtgc agagagctca tctctgacta tgctcctgac tgctttgtgc tcattgagat
     1441 gaatggcaag ctggtcaaaa ccaccattga ggaactcatc cccctcaagt acaccaggaa
     1501 ctaacacctg gtgcatgacc cgcaagcccg gtgcctgagc ggcccgcttc gagcagacat
     1561 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     1621 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     1681 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     1741 tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtcgaccga
     1801 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     1861 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     1921 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1981 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2041 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2101 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2161 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2221 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2281 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2341 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2401 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2461 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2521 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2581 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2641 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2701 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2761 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2821 aaatcaatct aaagtatata tgagtaaact tggtctgaca gcggccgcaa atgctaaacc
     2881 actgcagtgg ttaccagtgc ttgatcagtg aggcaccgat ctcagcgatc tgcctatttc
     2941 gttcgtccat agtggcctga ctccccgtcg tgtagatcac tacgattcgt gagggcttac
     3001 catcaggccc cagcgcagca atgatgccgc gagagccgcg ttcaccggcc cccgatttgt
     3061 cagcaatgaa ccagccagca gggagggccg agcgaagaag tggtcctgct actttgtccg
     3121 cctccatcca gtctatgagc tgctgtcgtg atgctagagt aagaagttcg ccagtgagta
     3181 gtttccgaag agttgtggcc attgctactg gcatcgtggt atcacgctcg tcgttcggta
     3241 tggcttcgtt caactctggt tcccagcggt caagccgggt cacatgatca cccatattat
     3301 gaagaaatgc agtcagctcc ttagggcctc cgatcgttgt cagaagtaag ttggccgcgg
     3361 tgttgtcgct catggtaatg gcagcactac acaattctct taccgtcatg ccatccgtaa
     3421 gatgcttttc cgtgaccggc gagtactcaa ccaagtcgtt ttgtgagtag tgtatacggc
     3481 gaccaagctg ctcttgcccg gcgtctatac gggacaacac cgcgccacat agcagtactt
     3541 tgaaagtgct catcatcggg aatcgttctt cggggcggaa agactcaagg atcttgccgc
     3601 tattgagatc cagttcgata tagcccactc ttgcacccag ttgatcttca gcatctttta
     3661 ctttcaccag cgtttcgggg tgtgcaaaaa caggcaagca aaatgccgca aagaagggaa
     3721 tgagtgcgac acgaaaatgt tggatgctca tactcgtcct ttttcaatat tattgaagca
     3781 tttatcagag ttactagtac gtctctcaag gataagtaag taatattaag gtacgggagg
     3841 tattggacag gccgc
//
