LOCUS       sequence-337981-        3899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal donor plasmid for CRISPitope method encoding mScarlet 
            fluorescent protein and puromycin resistance cassette [p.M1G].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3899)
  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 3899)
  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-PuroR [M1G]
FEATURES             Location/Qualifiers
     source          1..3899
                     /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..982
                     /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             986..1582
                     /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     1479..1499
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1607..1728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1618..1637)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1672..1691
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1976..1993)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2147..2735)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2227..2246)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2935..3726,3727..3795))
                     /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 gatccggaga gggcagagga agtctgctaa catgcggtga
      961 cgtcgaggag aatcctggcc cagggaccga gtacaagccc acggtgcgcc tcgccacccg
     1021 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     1081 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     1141 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     1201 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     1261 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     1321 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     1381 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     1441 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     1501 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     1561 gacccgcaag cccggtgcct gagcggcccg cttcgagcag acatgataag atacattgat
     1621 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     1681 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1741 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     1801 aacctctaca aatgtggtaa aatcgataag gatccgtcga ccgatgccct tgagagcctt
     1861 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac
     1921 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc
     1981 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2041 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2101 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2161 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2221 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2281 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2341 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2401 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2461 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2521 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     2581 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     2641 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     2701 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     2761 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     2821 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     2881 tatatgagta aacttggtct gacagcggcc gcaaatgcta aaccactgca gtggttacca
     2941 gtgcttgatc agtgaggcac cgatctcagc gatctgccta tttcgttcgt ccatagtggc
     3001 ctgactcccc gtcgtgtaga tcactacgat tcgtgagggc ttaccatcag gccccagcgc
     3061 agcaatgatg ccgcgagagc cgcgttcacc ggcccccgat ttgtcagcaa tgaaccagcc
     3121 agcagggagg gccgagcgaa gaagtggtcc tgctactttg tccgcctcca tccagtctat
     3181 gagctgctgt cgtgatgcta gagtaagaag ttcgccagtg agtagtttcc gaagagttgt
     3241 ggccattgct actggcatcg tggtatcacg ctcgtcgttc ggtatggctt cgttcaactc
     3301 tggttcccag cggtcaagcc gggtcacatg atcacccata ttatgaagaa atgcagtcag
     3361 ctccttaggg cctccgatcg ttgtcagaag taagttggcc gcggtgttgt cgctcatggt
     3421 aatggcagca ctacacaatt ctcttaccgt catgccatcc gtaagatgct tttccgtgac
     3481 cggcgagtac tcaaccaagt cgttttgtga gtagtgtata cggcgaccaa gctgctcttg
     3541 cccggcgtct atacgggaca acaccgcgcc acatagcagt actttgaaag tgctcatcat
     3601 cgggaatcgt tcttcggggc ggaaagactc aaggatcttg ccgctattga gatccagttc
     3661 gatatagccc actcttgcac ccagttgatc ttcagcatct tttactttca ccagcgtttc
     3721 ggggtgtgca aaaacaggca agcaaaatgc cgcaaagaag ggaatgagtg cgacacgaaa
     3781 atgttggatg ctcatactcg tcctttttca atattattga agcatttatc agagttacta
     3841 gtacgtctct caaggataag taagtaatat taaggtacgg gaggtattgg acaggccgc
//
