LOCUS       sequence-338126-        3837 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal donor plasmid for CRISPitope method encoding mScarlet 
            fluorescent protein, FLAG tag, Ovalbumin CD8+ T cell epitope [AA: 
            257-264 (SIINFEKL)] and blasticidin resistance cassette [p.M1G].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3837)
  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 3837)
  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-Ova-BlastR [M1G]
FEATURES             Location/Qualifiers
     source          1..3837
                     /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             929..952
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1010..1063
                     /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    1545..1666
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1556..1575)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1610..1629
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1914..1931)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2085..2673)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2165..2184)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2873..3664,3665..3733))
                     /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 ctgtacaagg gcagcggcga ctacaaggac gacgacgaca agcgcagcgg
      961 caagttcagc atcatcaact tcgagaagct ggtgtacaag ggatccggag agggcagagg
     1021 aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccagggaaga ccttcaacat
     1081 ctctcagcag gatctggagc tggtggaggt cgccactgag aagatcacca tgctctatga
     1141 ggacaacaag caccatgtcg gggcggccat caggaccaag actggggaga tcatctctgc
     1201 tgtccacatt gaggcctaca ttggcagggt cactgtctgt gctgaagcca ttgccattgg
     1261 gtctgctgtg agcaacgggc agaaggactt tgacaccatt gtggctgtca ggcaccccta
     1321 ctctgatgag gtggacagat ccatcagggt ggtcagcccc tgtggcatgt gcagagagct
     1381 catctctgac tatgctcctg actgctttgt gctcattgag atgaatggca agctggtcaa
     1441 aaccaccatt gaggaactca tccccctcaa gtacaccagg aactaacacc tggtgcatga
     1501 cccgcaagcc cggtgcctga gcggcccgct tcgagcagac atgataagat acattgatga
     1561 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1621 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     1681 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     1741 cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc gatgcccttg agagccttca
     1801 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     1861 tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc ttcctcgctc
     1921 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1981 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2041 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2101 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2161 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2221 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2281 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2341 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2401 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2461 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2521 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2581 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2641 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2701 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2761 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2821 tatgagtaaa cttggtctga cagcggccgc aaatgctaaa ccactgcagt ggttaccagt
     2881 gcttgatcag tgaggcaccg atctcagcga tctgcctatt tcgttcgtcc atagtggcct
     2941 gactccccgt cgtgtagatc actacgattc gtgagggctt accatcaggc cccagcgcag
     3001 caatgatgcc gcgagagccg cgttcaccgg cccccgattt gtcagcaatg aaccagccag
     3061 cagggagggc cgagcgaaga agtggtcctg ctactttgtc cgcctccatc cagtctatga
     3121 gctgctgtcg tgatgctaga gtaagaagtt cgccagtgag tagtttccga agagttgtgg
     3181 ccattgctac tggcatcgtg gtatcacgct cgtcgttcgg tatggcttcg ttcaactctg
     3241 gttcccagcg gtcaagccgg gtcacatgat cacccatatt atgaagaaat gcagtcagct
     3301 ccttagggcc tccgatcgtt gtcagaagta agttggccgc ggtgttgtcg ctcatggtaa
     3361 tggcagcact acacaattct cttaccgtca tgccatccgt aagatgcttt tccgtgaccg
     3421 gcgagtactc aaccaagtcg ttttgtgagt agtgtatacg gcgaccaagc tgctcttgcc
     3481 cggcgtctat acgggacaac accgcgccac atagcagtac tttgaaagtg ctcatcatcg
     3541 ggaatcgttc ttcggggcgg aaagactcaa ggatcttgcc gctattgaga tccagttcga
     3601 tatagcccac tcttgcaccc agttgatctt cagcatcttt tactttcacc agcgtttcgg
     3661 ggtgtgcaaa aacaggcaag caaaatgccg caaagaaggg aatgagtgcg acacgaaaat
     3721 gttggatgct catactcgtc ctttttcaat attattgaag catttatcag agttactagt
     3781 acgtctctca aggataagta agtaatatta aggtacggga ggtattggac aggccgc
//
