LOCUS       sequence-451249-        4197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous donor template for RELA knockout expressing EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4197)
  AUTHORS   Sato Yohei, Hanawa Yamato, Tsubota Akihito
  TITLE     CRISPR/Cas9-mediated RELA and RELC knockout in human regulatory T 
            cells abrogates FOXP3 expression and suppressive function.
  JOURNAL   Clinical Immunology Communications, Volume 6, 2024
REFERENCE   2  (bases 1 to 4197)
  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"; journalName: "Clinical 
            Immunology Communications, Volume 6, 2024"
COMMENT     Sequence Label: pUCmini-RELA-PGK-EGFP
FEATURES             Location/Qualifiers
     source          1..4197
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        489..999
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     918..936
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      1015..1024
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1021..1023,1024..1026,1027..1740)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1021..1023,1024..1026,1027..1737)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1066..1087)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1327..1346)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1674..1695
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1746..1857
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2448..3036)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2528..2547)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3207..3998,3999..4067))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3830..3849
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4068..4172)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcccaa tgatctcgag ttaggggaga acgcatctga ttcagtttcc tctctggagg
       61 gaggtggggc gtgccctccc tcaattttcc ttaagaaagt acaacaggcc ctgattcagc
      121 ttccctctgg agaagaagga ttccttaccc cgttttccct caagaagcag gtgtttctgg
      181 ttcctttcct ccacccctta gtttcaccgc aggttctagg gagcaggtcc tgactcagtt
      241 cccctgggag ggagcctggc ttctcctctc cgtggagagc agattcctcc ttgtgagaat
      301 gggcattggg tgtgtgctga ccctgatctc cctccccttt cagaactgtt ccccctcatc
      361 ttcccggcag gtaagtggcc cccgcggcgg tcggggaggt gctccctggg gtagggcagt
      421 gggggcagcc gcctaatggg gctgcggtgt cccctggcag agccagccca gggctctgga
      481 attcccacgg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc
      541 ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc
      601 ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg
      661 acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg
      721 tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca
      781 atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc ggggcgcgcc
      841 gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc
      901 ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc
      961 ggctccctcg ttgaccgaat caccgacctc tctccccagg gggatccacc ggtcgccacc
     1021 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1081 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1141 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1201 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1261 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1321 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1381 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1441 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1501 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1561 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1621 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1681 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     1741 gtcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     1801 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcatc
     1861 gatgccccta tgtggagatc attgagcagc ccaagcagcg gggcatgcgc ttccgctaca
     1921 agtgcgaggg gcgctccgcg ggcagcatcc caggcgagag gagcacagat accaccaaga
     1981 cccaccccac catcaaggtc agcactgcgc cagggtgtgc ggggaaggga ctgtggaacc
     2041 ccagccctct gtgtttggga gtcaggtttg gatgtctggg cctctgtact ttggacagat
     2101 caatggctac acaggaccag ggacagtgcg catctccctg gtcaccaagg accctcctca
     2161 ccggcctcac ccccacgagc ttgtaggaaa ggactgccgg gatggcttct atgaggctga
     2221 gctctgcccg gaccgctgca tccacaggtg aggtccctgc aggctaggag gcccagcatg
     2281 aggggacagg aggcgggcta tgaagtagcc gctacagtgt tactcctttc tggacccagg
     2341 gcagaattcc tcactccttg cccaactcag gctagcatcg gaaagaacat gtgagcaaaa
     2401 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2461 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2521 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2581 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2641 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2701 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2761 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2821 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2881 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2941 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3001 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3061 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3121 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3181 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3241 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3301 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3361 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3421 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3481 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3541 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3601 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3661 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3721 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3781 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3841 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3901 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3961 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4021 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4081 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4141 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacct
//
