LOCUS       sequence-338124-        3843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal donor plasmid for CRISPitope method encoding mScarlet 
            fluorescent protein, FLAG tag, HSV-1 glycoprotein B CD8+ T cell 
            epitope [AA: 498-505 (SSIEFARL)] and blasticidin resistance cassette
            [p..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3843)
  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 3843)
  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-gB-BlastR [M1G]
FEATURES             Location/Qualifiers
     source          1..3843
                     /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             1016..1069
                     /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    1551..1672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1562..1581)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1616..1635
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1920..1937)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2091..2679)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2171..2190)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2879..3670,3671..3739))
                     /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 ctgagcagca tcgagttcgc cagactggtg tacaagggat ccggagaggg
     1021 cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag ggaagacctt
     1081 caacatctct cagcaggatc tggagctggt ggaggtcgcc actgagaaga tcaccatgct
     1141 ctatgaggac aacaagcacc atgtcggggc ggccatcagg accaagactg gggagatcat
     1201 ctctgctgtc cacattgagg cctacattgg cagggtcact gtctgtgctg aagccattgc
     1261 cattgggtct gctgtgagca acgggcagaa ggactttgac accattgtgg ctgtcaggca
     1321 cccctactct gatgaggtgg acagatccat cagggtggtc agcccctgtg gcatgtgcag
     1381 agagctcatc tctgactatg ctcctgactg ctttgtgctc attgagatga atggcaagct
     1441 ggtcaaaacc accattgagg aactcatccc cctcaagtac accaggaact aacacctggt
     1501 gcatgacccg caagcccggt gcctgagcgg cccgcttcga gcagacatga taagatacat
     1561 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     1621 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     1681 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     1741 gtaaaacctc tacaaatgtg gtaaaatcga taaggatccg tcgaccgatg cccttgagag
     1801 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     1861 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc
     1921 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     1981 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2041 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2101 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2161 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2221 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2281 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2341 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2401 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2461 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2521 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2581 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2641 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2701 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2761 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2821 agtatatatg agtaaacttg gtctgacagc ggccgcaaat gctaaaccac tgcagtggtt
     2881 accagtgctt gatcagtgag gcaccgatct cagcgatctg cctatttcgt tcgtccatag
     2941 tggcctgact ccccgtcgtg tagatcacta cgattcgtga gggcttacca tcaggcccca
     3001 gcgcagcaat gatgccgcga gagccgcgtt caccggcccc cgatttgtca gcaatgaacc
     3061 agccagcagg gagggccgag cgaagaagtg gtcctgctac tttgtccgcc tccatccagt
     3121 ctatgagctg ctgtcgtgat gctagagtaa gaagttcgcc agtgagtagt ttccgaagag
     3181 ttgtggccat tgctactggc atcgtggtat cacgctcgtc gttcggtatg gcttcgttca
     3241 actctggttc ccagcggtca agccgggtca catgatcacc catattatga agaaatgcag
     3301 tcagctcctt agggcctccg atcgttgtca gaagtaagtt ggccgcggtg ttgtcgctca
     3361 tggtaatggc agcactacac aattctctta ccgtcatgcc atccgtaaga tgcttttccg
     3421 tgaccggcga gtactcaacc aagtcgtttt gtgagtagtg tatacggcga ccaagctgct
     3481 cttgcccggc gtctatacgg gacaacaccg cgccacatag cagtactttg aaagtgctca
     3541 tcatcgggaa tcgttcttcg gggcggaaag actcaaggat cttgccgcta ttgagatcca
     3601 gttcgatata gcccactctt gcacccagtt gatcttcagc atcttttact ttcaccagcg
     3661 tttcggggtg tgcaaaaaca ggcaagcaaa atgccgcaaa gaagggaatg agtgcgacac
     3721 gaaaatgttg gatgctcata ctcgtccttt ttcaatatta ttgaagcatt tatcagagtt
     3781 actagtacgt ctctcaagga taagtaagta atattaaggt acgggaggta ttggacaggc
     3841 cgc
//
