LOCUS       sequence-452314-        4794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous donor template for RELC knockout expressing tdTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4794)
  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 4794)
  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-tdTomato
FEATURES             Location/Qualifiers
     source          1..4794
                     /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             963..2393
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     polyA_signal    2399..2510
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3045..3633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3125..3144)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3804..4595,4596..4664))
                     /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     4427..4446
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4665..4769)
                     /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 gaggtcatca aagagttcat gcgcttcaag gtgcgcatgg
     1021 agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1081 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
     1141 acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca
     1201 tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1261 tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga
     1321 tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga
     1381 agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg
     1441 gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga
     1501 ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc
     1561 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg
     1621 gccgccacca cctgttcctg gggcatggca ccggcagcac cggcagcggc agctccggca
     1681 ccgcctcctc cgaggacaac aacatggccg tcatcaaaga gttcatgcgc ttcaaggtgc
     1741 gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1801 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg
     1861 cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg aagcaccccg
     1921 ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     1981 tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg caggacggca
     2041 cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc cccgtaatgc
     2101 agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc
     2161 tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccactac ctggtggagt
     2221 tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac tacgtggaca
     2281 ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgct
     2341 ccgagggccg ccaccacctg ttcctgtacg gcatggacga gctgtacaag taggtcgact
     2401 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2461 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc atcgatccca
     2521 gccagagggg aatgcgtttt agatacaaat gtgaagggcg atcagcaggc agcattccag
     2581 gggagcacag cacagacaac aaccgaacat acccttctat ccaggtaata gacccttctt
     2641 ctgtgtctat ctcactatta gttgcttcat atactagcta tagcaattat tttaaagggc
     2701 cagtttcttc acagtcatct ccacaggttt atctttttct tttttccttt ttttttttaa
     2761 acggatctgt caaaaagaca tctattagat agatgtctaa ggacatttct taaatataaa
     2821 atggagtctt ccccactgca gatagttcta tggtgcttgt gtattaacct aagtagagcg
     2881 actcaatgtt gagagtacac tggcaacttt tggatcatgt tctttttcat ggaacacaga
     2941 gataataagg aaaacccatt ctctgcagct agcatcggaa agaacatgtg agcaaaaggc
     3001 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3061 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3121 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3181 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3241 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3301 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3361 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3421 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3481 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3541 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3601 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3661 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3721 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3781 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3841 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3901 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3961 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4021 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4081 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4141 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4201 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4261 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4321 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4381 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4441 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4501 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4561 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4621 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4681 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4741 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acct
//
