LOCUS       sequence-337985-        3986 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 puromycin resistance cassette 
            [p.M1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3986)
  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 3986)
  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-PuroR [M1G]
FEATURES             Location/Qualifiers
     source          1..3986
                     /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"
     CDS             1073..1669
                     /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     1566..1586
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1694..1815
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1705..1724)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1759..1778
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2063..2080)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2234..2822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2314..2333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3022..3813,3814..3882))
                     /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 ggaccgagta
     1081 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     1141 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     1201 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     1261 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     1321 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     1381 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     1441 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     1501 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     1561 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     1621 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag cggcccgctt
     1681 cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     1741 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     1801 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     1861 gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cgataaggat
     1921 ccgtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     1981 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     2041 gccggcagcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2101 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2161 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2221 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2281 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2341 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2401 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2461 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2521 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2581 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2641 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2701 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2761 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2821 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2881 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2941 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agcggccgca
     3001 aatgctaaac cactgcagtg gttaccagtg cttgatcagt gaggcaccga tctcagcgat
     3061 ctgcctattt cgttcgtcca tagtggcctg actccccgtc gtgtagatca ctacgattcg
     3121 tgagggctta ccatcaggcc ccagcgcagc aatgatgccg cgagagccgc gttcaccggc
     3181 ccccgatttg tcagcaatga accagccagc agggagggcc gagcgaagaa gtggtcctgc
     3241 tactttgtcc gcctccatcc agtctatgag ctgctgtcgt gatgctagag taagaagttc
     3301 gccagtgagt agtttccgaa gagttgtggc cattgctact ggcatcgtgg tatcacgctc
     3361 gtcgttcggt atggcttcgt tcaactctgg ttcccagcgg tcaagccggg tcacatgatc
     3421 acccatatta tgaagaaatg cagtcagctc cttagggcct ccgatcgttg tcagaagtaa
     3481 gttggccgcg gtgttgtcgc tcatggtaat ggcagcacta cacaattctc ttaccgtcat
     3541 gccatccgta agatgctttt ccgtgaccgg cgagtactca accaagtcgt tttgtgagta
     3601 gtgtatacgg cgaccaagct gctcttgccc ggcgtctata cgggacaaca ccgcgccaca
     3661 tagcagtact ttgaaagtgc tcatcatcgg gaatcgttct tcggggcgga aagactcaag
     3721 gatcttgccg ctattgagat ccagttcgat atagcccact cttgcaccca gttgatcttc
     3781 agcatctttt actttcacca gcgtttcggg gtgtgcaaaa acaggcaagc aaaatgccgc
     3841 aaagaaggga atgagtgcga cacgaaaatg ttggatgctc atactcgtcc tttttcaata
     3901 ttattgaagc atttatcaga gttactagta cgtctctcaa ggataagtaa gtaatattaa
     3961 ggtacgggag gtattggaca ggccgc
//
