LOCUS       sequence-338044-        3980 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 puromycin resistance cassette [p.M1G].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3980)
  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 3980)
  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-PuroR [M1G]
FEATURES             Location/Qualifiers
     source          1..3980
                     /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"
     CDS             1067..1663
                     /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     1560..1580
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1688..1809
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1699..1718)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1753..1772
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2057..2074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2228..2816)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2308..2327)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3016..3807,3808..3876))
                     /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 ccagggaccg agtacaagcc
     1081 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     1141 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     1201 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     1261 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     1321 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     1381 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     1441 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     1501 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     1561 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     1621 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgagcggccc gcttcgagca
     1681 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     1741 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     1801 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     1861 gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatccgtcg
     1921 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
     1981 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
     2041 agcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2101 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2161 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2221 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2281 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2341 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2401 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2461 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2521 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2581 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2641 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2701 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2761 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2821 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2881 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2941 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagcggc cgcaaatgct
     3001 aaaccactgc agtggttacc agtgcttgat cagtgaggca ccgatctcag cgatctgcct
     3061 atttcgttcg tccatagtgg cctgactccc cgtcgtgtag atcactacga ttcgtgaggg
     3121 cttaccatca ggccccagcg cagcaatgat gccgcgagag ccgcgttcac cggcccccga
     3181 tttgtcagca atgaaccagc cagcagggag ggccgagcga agaagtggtc ctgctacttt
     3241 gtccgcctcc atccagtcta tgagctgctg tcgtgatgct agagtaagaa gttcgccagt
     3301 gagtagtttc cgaagagttg tggccattgc tactggcatc gtggtatcac gctcgtcgtt
     3361 cggtatggct tcgttcaact ctggttccca gcggtcaagc cgggtcacat gatcacccat
     3421 attatgaaga aatgcagtca gctccttagg gcctccgatc gttgtcagaa gtaagttggc
     3481 cgcggtgttg tcgctcatgg taatggcagc actacacaat tctcttaccg tcatgccatc
     3541 cgtaagatgc ttttccgtga ccggcgagta ctcaaccaag tcgttttgtg agtagtgtat
     3601 acggcgacca agctgctctt gcccggcgtc tatacgggac aacaccgcgc cacatagcag
     3661 tactttgaaa gtgctcatca tcgggaatcg ttcttcgggg cggaaagact caaggatctt
     3721 gccgctattg agatccagtt cgatatagcc cactcttgca cccagttgat cttcagcatc
     3781 ttttactttc accagcgttt cggggtgtgc aaaaacaggc aagcaaaatg ccgcaaagaa
     3841 gggaatgagt gcgacacgaa aatgttggat gctcatactc gtcctttttc aatattattg
     3901 aagcatttat cagagttact agtacgtctc tcaaggataa gtaagtaata ttaaggtacg
     3961 ggaggtattg gacaggccgc
//
