LOCUS       sequence-333480-        3765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal donor plasmid for CRISPitope method encoding mNeon 
            fluorescent protein and blasticidin resistance cassette [p.M1G].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3765)
  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 3765)
  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-mNeon-BlastR [M1G]
FEATURES             Location/Qualifiers
     source          1..3765
                     /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..928
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEEL
                     NLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
                     SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTF
                     KWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKA
                     FTDVMGMDELYK"
     regulatory      476..485
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             938..991
                     /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    1473..1594
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1484..1503)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1538..1557
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1842..1859)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2013..2601)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2093..2112)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2801..3592,3593..3661))
                     /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 ggaggataac gcctctctcc cagcgacaca tgagttacac atctttggct ccatcaacgg
      301 tgtggacttt gacatggtgg gtcagggcac cggcaatcca aatgatggtt atgaggagtt
      361 aaacctgaag tccaccaagg gtgacctcca gttctccccc tggattctgg tccctcatat
      421 cgggtatggc ttccatcagt acctgcccta ccctgacggg atgtcgcctt tccaggccgc
      481 catggtagat ggctccggat accaagtcca tcgcacaatg cagtttgaag atggtgcctc
      541 ccttactgtt aactaccgct acacctacga gggaagccac atcaaaggag aggcccaggt
      601 gaaggggact ggtttccctg ctgacggtcc tgtgatgacc aactcgctga ccgctgcgga
      661 ctggtgcagg tcgaagaaga cttaccccaa cgacaaaacc atcatcagta cctttaagtg
      721 gagttacacc actggaaatg gcaagcgcta ccggagcact gcgcggacca cctacacctt
      781 tgccaagcca atggcggcta actatctgaa gaaccagccg atgtacgtgt tccgtaagac
      841 ggagctcaag cactccaaga ccgagctcaa cttcaaggag tggcaaaagg cctttaccga
      901 tgtgatgggc atggacgagc tgtacaaggg atccggagag ggcagaggaa gtctgctaac
      961 atgcggtgac gtcgaggaga atcctggccc agggaagacc ttcaacatct ctcagcagga
     1021 tctggagctg gtggaggtcg ccactgagaa gatcaccatg ctctatgagg acaacaagca
     1081 ccatgtcggg gcggccatca ggaccaagac tggggagatc atctctgctg tccacattga
     1141 ggcctacatt ggcagggtca ctgtctgtgc tgaagccatt gccattgggt ctgctgtgag
     1201 caacgggcag aaggactttg acaccattgt ggctgtcagg cacccctact ctgatgaggt
     1261 ggacagatcc atcagggtgg tcagcccctg tggcatgtgc agagagctca tctctgacta
     1321 tgctcctgac tgctttgtgc tcattgagat gaatggcaag ctggtcaaaa ccaccattga
     1381 ggaactcatc cccctcaagt acaccaggaa ctaacacctg gtgcatgacc cgcaagcccg
     1441 gtgcctgagc ggcccgcttc gagcagacat gataagatac attgatgagt ttggacaaac
     1501 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     1561 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     1621 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     1681 tggtaaaatc gataaggatc cgtcgaccga tgcccttgag agccttcaac ccagtcagct
     1741 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     1801 tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg
     1861 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     1921 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     1981 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2041 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2101 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2161 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2221 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2281 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2341 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     2401 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     2461 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     2521 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     2581 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     2641 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     2701 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     2761 tggtctgaca gcggccgcaa atgctaaacc actgcagtgg ttaccagtgc ttgatcagtg
     2821 aggcaccgat ctcagcgatc tgcctatttc gttcgtccat agtggcctga ctccccgtcg
     2881 tgtagatcac tacgattcgt gagggcttac catcaggccc cagcgcagca atgatgccgc
     2941 gagagccgcg ttcaccggcc cccgatttgt cagcaatgaa ccagccagca gggagggccg
     3001 agcgaagaag tggtcctgct actttgtccg cctccatcca gtctatgagc tgctgtcgtg
     3061 atgctagagt aagaagttcg ccagtgagta gtttccgaag agttgtggcc attgctactg
     3121 gcatcgtggt atcacgctcg tcgttcggta tggcttcgtt caactctggt tcccagcggt
     3181 caagccgggt cacatgatca cccatattat gaagaaatgc agtcagctcc ttagggcctc
     3241 cgatcgttgt cagaagtaag ttggccgcgg tgttgtcgct catggtaatg gcagcactac
     3301 acaattctct taccgtcatg ccatccgtaa gatgcttttc cgtgaccggc gagtactcaa
     3361 ccaagtcgtt ttgtgagtag tgtatacggc gaccaagctg ctcttgcccg gcgtctatac
     3421 gggacaacac cgcgccacat agcagtactt tgaaagtgct catcatcggg aatcgttctt
     3481 cggggcggaa agactcaagg atcttgccgc tattgagatc cagttcgata tagcccactc
     3541 ttgcacccag ttgatcttca gcatctttta ctttcaccag cgtttcgggg tgtgcaaaaa
     3601 caggcaagca aaatgccgca aagaagggaa tgagtgcgac acgaaaatgt tggatgctca
     3661 tactcgtcct ttttcaatat tattgaagca tttatcagag ttactagtac gtctctcaag
     3721 gataagtaag taatattaag gtacgggagg tattggacag gccgc
//
