LOCUS       sequence-452318-        4083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous donor template for RELC knockout expressing EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4083)
  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 4083)
  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-RELC-PGK-EGFP
FEATURES             Location/Qualifiers
     source          1..4083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        431..941
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     860..878
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      957..966
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(963..965,966..968,969..1682)
                     /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(963..965,966..968,969..1679)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1008..1029)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1269..1288)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1616..1637
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1688..1799
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2334..2922)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2414..2433)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3093..3884,3885..3953))
                     /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     3716..3735
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3954..4058)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcccaa tgatctcgag acccttcttg gggaatcttg ggggaaaccc ttctgctagg
       61 tctcacgtgg gaggcagata tctctgaagt ggttatctca gaacttcata ttgacgttag
      121 ctatagatat taagctataa agtgctaacc agtcaatatg gtttgaatga cttgttttga
      181 cactacatag ggactcaatt tgaatgggaa aaaaaggtgc ctctcccagc caatctccaa
      241 gataacattt cggtcttgtt atttaatgtt aggaggaaaa aaatctttgt tttagtctgc
      301 tgttataaaa aaaacagaat gttaaaatcc attattaatt gctatattaa tctcactgac
      361 ctcctccttt ttaaaaactt ttcaggtgcg tataacccgt atatagagat aattgaacaa
      421 gaattcccac ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac
      481 gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat
      541 tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg
      601 cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga
      661 cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag
      721 caatggcagc gcgccgaccg cgatgggctg tggccaatag cggctgctca gcggggcgcg
      781 ccgagagcag cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg
      841 ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag
      901 tcggctccct cgttgaccga atcaccgacc tctctcccca gggggatcca ccggtcgcca
      961 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1021 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1081 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1141 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1201 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1261 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1321 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1381 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1441 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1501 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1561 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1621 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1681 aagtcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1741 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatca
     1801 tcgatcccag ccagagggga atgcgtttta gatacaaatg tgaagggcga tcagcaggca
     1861 gcattccagg ggagcacagc acagacaaca accgaacata cccttctatc caggtaatag
     1921 acccttcttc tgtgtctatc tcactattag ttgcttcata tactagctat agcaattatt
     1981 ttaaagggcc agtttcttca cagtcatctc cacaggttta tctttttctt ttttcctttt
     2041 tttttttaaa cggatctgtc aaaaagacat ctattagata gatgtctaag gacatttctt
     2101 aaatataaaa tggagtcttc cccactgcag atagttctat ggtgcttgtg tattaaccta
     2161 agtagagcga ctcaatgttg agagtacact ggcaactttt ggatcatgtt ctttttcatg
     2221 gaacacagag ataataagga aaacccattc tctgcagcta gcatcggaaa gaacatgtga
     2281 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2341 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2401 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2461 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2521 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2581 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2641 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2701 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2761 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2821 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2881 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2941 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3001 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3061 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3121 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3181 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3241 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3301 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3361 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3421 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3481 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3541 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3601 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3661 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3721 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3781 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3841 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3901 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3961 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4021 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4081 cct
//
