LOCUS       sequence-337973-        3997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal donor plasmid for CRISPitope method encoding mNeon 
            fluorescent protein, FLAG tag, murine gp100 CD8+ T cell epitope [AA:
            25-33 (EGSRNQDWL)] and puromycin resistance cassette [p.M1G].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3997)
  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 3997)
  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-mgp100-PuroR [M1G]
FEATURES             Location/Qualifiers
     source          1..3997
                     /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             226..927
                     /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      475..484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             940..963
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1027..1080
                     /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             1084..1680
                     /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     1577..1597
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1705..1826
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1716..1735)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1770..1789
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2074..2091)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2245..2833)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2325..2344)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3033..3824,3825..3893))
                     /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 aaagcggggg gtctggtggc agtggagggg gatccgtgag caagggcgag
      241 gaggataacg cctctctccc agcgacacat gagttacaca tctttggctc catcaacggt
      301 gtggactttg acatggtggg tcagggcacc ggcaatccaa atgatggtta tgaggagtta
      361 aacctgaagt ccaccaaggg tgacctccag ttctccccct ggattctggt ccctcatatc
      421 gggtatggct tccatcagta cctgccctac cctgacggga tgtcgccttt ccaggccgcc
      481 atggtagatg gctccggata ccaagtccat cgcacaatgc agtttgaaga tggtgcctcc
      541 cttactgtta actaccgcta cacctacgag ggaagccaca tcaaaggaga ggcccaggtg
      601 aaggggactg gtttccctgc tgacggtcct gtgatgacca actcgctgac cgctgcggac
      661 tggtgcaggt cgaagaagac ttaccccaac gacaaaacca tcatcagtac ctttaagtgg
      721 agttacacca ctggaaatgg caagcgctac cggagcactg cgcggaccac ctacaccttt
      781 gccaagccaa tggcggctaa ctatctgaag aaccagccga tgtacgtgtt ccgtaagacg
      841 gagctcaagc actccaagac cgagctcaac ttcaaggagt ggcaaaaggc ctttaccgat
      901 gtgatgggca tggacgagct gtacaagagc ggctccgccg attacaagga tgacgacgac
      961 aaacgatcgg ggcctaggct ggaagggtcg cgcaaccagg attggctggt gtacaaggga
     1021 tccggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca
     1081 gggaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     1141 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     1201 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     1261 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     1321 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     1381 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     1441 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     1501 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     1561 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     1621 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     1681 gcggcccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta
     1741 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     1801 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     1861 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa
     1921 tcgataagga tccgtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     1981 tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc
     2041 gtaggacagg tgccggcagc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2101 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     2161 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2221 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2281 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2341 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2401 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2461 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2521 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     2581 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     2641 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     2701 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     2761 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     2821 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     2881 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     2941 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3001 cagcggccgc aaatgctaaa ccactgcagt ggttaccagt gcttgatcag tgaggcaccg
     3061 atctcagcga tctgcctatt tcgttcgtcc atagtggcct gactccccgt cgtgtagatc
     3121 actacgattc gtgagggctt accatcaggc cccagcgcag caatgatgcc gcgagagccg
     3181 cgttcaccgg cccccgattt gtcagcaatg aaccagccag cagggagggc cgagcgaaga
     3241 agtggtcctg ctactttgtc cgcctccatc cagtctatga gctgctgtcg tgatgctaga
     3301 gtaagaagtt cgccagtgag tagtttccga agagttgtgg ccattgctac tggcatcgtg
     3361 gtatcacgct cgtcgttcgg tatggcttcg ttcaactctg gttcccagcg gtcaagccgg
     3421 gtcacatgat cacccatatt atgaagaaat gcagtcagct ccttagggcc tccgatcgtt
     3481 gtcagaagta agttggccgc ggtgttgtcg ctcatggtaa tggcagcact acacaattct
     3541 cttaccgtca tgccatccgt aagatgcttt tccgtgaccg gcgagtactc aaccaagtcg
     3601 ttttgtgagt agtgtatacg gcgaccaagc tgctcttgcc cggcgtctat acgggacaac
     3661 accgcgccac atagcagtac tttgaaagtg ctcatcatcg ggaatcgttc ttcggggcgg
     3721 aaagactcaa ggatcttgcc gctattgaga tccagttcga tatagcccac tcttgcaccc
     3781 agttgatctt cagcatcttt tactttcacc agcgtttcgg ggtgtgcaaa aacaggcaag
     3841 caaaatgccg caaagaaggg aatgagtgcg acacgaaaat gttggatgct catactcgtc
     3901 ctttttcaat attattgaag catttatcag agttactagt acgtctctca aggataagta
     3961 agtaatatta aggtacggga ggtattggac aggccgc
//
