LOCUS       sequence-447537-        4795 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 4795)
  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 4795)
  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..4795
                     /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    2400..2511
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3046..3634)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3126..3145)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3805..4596,4597..4665))
                     /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     4428..4447
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4666..4770)
                     /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 taggtcgacc
     2401 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2461 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat catcgatccc
     2521 agccagaggg gaatgcgttt tagatacaaa tgtgaagggc gatcagcagg cagcattcca
     2581 ggggagcaca gcacagacaa caaccgaaca tacccttcta tccaggtaat agacccttct
     2641 tctgtgtcta tctcactatt agttgcttca tatactagct atagcaatta ttttaaaggg
     2701 ccagtttctt cacagtcatc tccacaggtt tatctttttc ttttttcctt ttttttttta
     2761 aacggatctg tcaaaaagac atctattaga tagatgtcta aggacatttc ttaaatataa
     2821 aatggagtct tccccactgc agatagttct atggtgcttg tgtattaacc taagtagagc
     2881 gactcaatgt tgagagtaca ctggcaactt ttggatcatg ttctttttca tggaacacag
     2941 agataataag gaaaacccat tctctgcagc tagcatcgga aagaacatgt gagcaaaagg
     3001 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3061 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3121 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3181 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3241 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3301 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3361 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3421 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3481 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3541 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3601 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3661 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3721 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3781 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3841 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3901 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3961 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4021 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4081 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4141 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4201 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4261 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4321 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4381 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4441 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4501 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4561 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4621 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4681 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4741 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacct
//
