LOCUS       sequence-338123-        3846 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal donor plasmid for CRISPitope method encoding mScarlet 
            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 3846)
  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 3846)
  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-mScarlet-F-hgp100-BlastR [M1G]
FEATURES             Location/Qualifiers
     source          1..3846
                     /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..919
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(357..375)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     625..644
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             932..955
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1019..1072
                     /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    1554..1675
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1565..1584)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1619..1638
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1923..1940)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2094..2682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2174..2193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2882..3673,3674..3742))
                     /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 ggcagtgatc aaggagttca tgcggttcaa ggtgcacatg gagggctcca tgaacggcca
      301 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
      361 gctgaaggtg accaagggtg gccccctgcc cttctcctgg gacatcctgt cccctcagtt
      421 catgtacggc tccagggcct tcaccaagca ccccgccgac atccccgact actataagca
      481 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgccgt
      541 gaccgtgacc caggacacct ccctggagga cggcaccctg atctacaagg tgaagctccg
      601 cggcaccaac ttccctcctg acggccccgt aatgcagaag aagacaatgg gctgggaagc
      661 gtccaccgag cggttgtacc ccgaggacgg cgtgctgaag ggcgacatta agatggccct
      721 gcgcctgaag gacggcggcc gctacctggc ggacttcaag accacctaca aggccaagaa
      781 gcccgtgcag atgcccggcg cctacaacgt cgaccgcaag ttggacatca cctcccacaa
      841 cgaggactac accgtggtgg aacagtacga acgctccgag ggccgccact ccaccggcgg
      901 catggacgag ctgtacaaga gcggctccgc cgattacaag gatgacgacg acaaacgatc
      961 ggggcctagg ctgaaagtgc cgcgcaacca ggattggctg gtgtacaagg gatccggaga
     1021 gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagggaagac
     1081 cttcaacatc tctcagcagg atctggagct ggtggaggtc gccactgaga agatcaccat
     1141 gctctatgag gacaacaagc accatgtcgg ggcggccatc aggaccaaga ctggggagat
     1201 catctctgct gtccacattg aggcctacat tggcagggtc actgtctgtg ctgaagccat
     1261 tgccattggg tctgctgtga gcaacgggca gaaggacttt gacaccattg tggctgtcag
     1321 gcacccctac tctgatgagg tggacagatc catcagggtg gtcagcccct gtggcatgtg
     1381 cagagagctc atctctgact atgctcctga ctgctttgtg ctcattgaga tgaatggcaa
     1441 gctggtcaaa accaccattg aggaactcat ccccctcaag tacaccagga actaacacct
     1501 ggtgcatgac ccgcaagccc ggtgcctgag cggcccgctt cgagcagaca tgataagata
     1561 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     1621 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     1681 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
     1741 caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccgtcgaccg atgcccttga
     1801 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     1861 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctcttccgct
     1921 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1981 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2041 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2101 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2161 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2221 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2281 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2341 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2401 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2461 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2521 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2581 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2641 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2701 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2761 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2821 taaagtatat atgagtaaac ttggtctgac agcggccgca aatgctaaac cactgcagtg
     2881 gttaccagtg cttgatcagt gaggcaccga tctcagcgat ctgcctattt cgttcgtcca
     2941 tagtggcctg actccccgtc gtgtagatca ctacgattcg tgagggctta ccatcaggcc
     3001 ccagcgcagc aatgatgccg cgagagccgc gttcaccggc ccccgatttg tcagcaatga
     3061 accagccagc agggagggcc gagcgaagaa gtggtcctgc tactttgtcc gcctccatcc
     3121 agtctatgag ctgctgtcgt gatgctagag taagaagttc gccagtgagt agtttccgaa
     3181 gagttgtggc cattgctact ggcatcgtgg tatcacgctc gtcgttcggt atggcttcgt
     3241 tcaactctgg ttcccagcgg tcaagccggg tcacatgatc acccatatta tgaagaaatg
     3301 cagtcagctc cttagggcct ccgatcgttg tcagaagtaa gttggccgcg gtgttgtcgc
     3361 tcatggtaat ggcagcacta cacaattctc ttaccgtcat gccatccgta agatgctttt
     3421 ccgtgaccgg cgagtactca accaagtcgt tttgtgagta gtgtatacgg cgaccaagct
     3481 gctcttgccc ggcgtctata cgggacaaca ccgcgccaca tagcagtact ttgaaagtgc
     3541 tcatcatcgg gaatcgttct tcggggcgga aagactcaag gatcttgccg ctattgagat
     3601 ccagttcgat atagcccact cttgcaccca gttgatcttc agcatctttt actttcacca
     3661 gcgtttcggg gtgtgcaaaa acaggcaagc aaaatgccgc aaagaaggga atgagtgcga
     3721 cacgaaaatg ttggatgctc atactcgtcc tttttcaata ttattgaagc atttatcaga
     3781 gttactagta cgtctctcaa ggataagtaa gtaatattaa ggtacgggag gtattggaca
     3841 ggccgc
//
