LOCUS       sequence-451172-        4908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous donor template for RELA knockout expressing tdTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4908)
  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 4908)
  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-tdTomato
FEATURES             Location/Qualifiers
     source          1..4908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        488..998
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     917..935
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      1014..1023
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1020..2450
                     /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    2456..2567
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3159..3747)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3239..3258)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3918..4709,4710..4778))
                     /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     4541..4560
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4779..4883)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcccaa gatctcgagt taggggagaa cgcatctgat tcagtttcct ctctggaggg
       61 aggtggggcg tgccctccct cagttttcct taagaaagta caacaggccc tgattcagct
      121 tccctctgga gaagaaggat tccttacccc gttttccctc aagaagcagg tgtttctggt
      181 tcctttcctc caccccttag tttcaccgca ggttctaggg agcaggtcct gactcagttc
      241 ccctgggagg gagcctggct tctcctctcc gtggagagca gattcctcct tgtgagaatg
      301 ggcattgggt gtgtgctgac cctgatctcc ctcccctttc agaactgttc cccctcatct
      361 tcccggcagg taagtggccc ccgcggcggt cggggaggtg ctccctgggg tagggcagtg
      421 ggggcagccg cctaatgggg ctgcggtgtc ccctggcaga gccagcccag ggctctggaa
      481 ttcccacggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg
      541 gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct
      601 tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga
      661 cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt
      721 gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa
      781 tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagcg gggcgcgccg
      841 agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc
      901 tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg
      961 gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatccaccg gtcgccacca
     1021 tggtgagcaa gggcgaggag gtcatcaaag agttcatgcg cttcaaggtg cgcatggagg
     1081 gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     1141 gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca
     1201 tcctgtcccc ccagttcatg tacggctcca aggcgtacgt gaagcacccc gccgacatcc
     1261 ccgattacaa gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     1321 aggacggcgg tctggtgacc gtgacccagg actcctccct gcaggacggc acgctgatct
     1381 acaaggtgaa gatgcgcggc accaacttcc cccccgacgg ccccgtaatg cagaagaaga
     1441 ccatgggctg ggaggcctcc accgagcgcc tgtacccccg cgacggcgtg ctgaagggcg
     1501 agatccacca ggccctgaag ctgaaggacg gcggccacta cctggtggag ttcaagacca
     1561 tctacatggc caagaagccc gtgcaactgc ccggctacta ctacgtggac accaagctgg
     1621 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc tccgagggcc
     1681 gccaccacct gttcctgggg catggcaccg gcagcaccgg cagcggcagc tccggcaccg
     1741 cctcctccga ggacaacaac atggccgtca tcaaagagtt catgcgcttc aaggtgcgca
     1801 tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     1861 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg cccttcgcct
     1921 gggacatcct gtccccccag ttcatgtacg gctccaaggc gtacgtgaag caccccgccg
     1981 acatccccga ttacaagaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     2041 acttcgagga cggcggtctg gtgaccgtga cccaggactc ctccctgcag gacggcacgc
     2101 tgatctacaa ggtgaagatg cgcggcacca acttcccccc cgacggcccc gtaatgcaga
     2161 agaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac ggcgtgctga
     2221 agggcgagat ccaccaggcc ctgaagctga aggacggcgg ccactacctg gtggagttca
     2281 agaccatcta catggccaag aagcccgtgc aactgcccgg ctactactac gtggacacca
     2341 agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gagcgctccg
     2401 agggccgcca ccacctgttc ctgtacggca tggacgagct gtacaagtag gtcgactgtg
     2461 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2521 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcatc gatgccccta
     2581 tgtggagatc attgagcagc ccaagcagcg gggcatgcgc ttccgctaca agtgcgaggg
     2641 gcgctccgcg ggcagcatcc caggcgagag gagcacagat accaccaaga cccaccccac
     2701 catcaaggtc agcactgcgc cagggtgtgc ggggaaggga ctgtggaacc ccagccctct
     2761 gtgtttggga gtcaggtttg gatgtctggg cctctgtact ttggacagat caatggctac
     2821 acaggaccag ggacagtgcg catctccctg gtcaccaagg accctcctca ccggcctcac
     2881 ccccacgagc ttgtaggaaa ggactgccgg gatggcttct atgaggctga gctctgcccg
     2941 gaccgctgca tccacaggtg aggtccctgc aggctaggag gcccagcatg aggggacagg
     3001 aggcgggcta tgaagtagcc gctacagtgt tactcctttc tggacccagg gcagaattcc
     3061 tcactccttg ccctgactca ggctagcatc ggaaagaaca tgtgagcaaa aggccagcaa
     3121 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3181 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3241 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3301 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3361 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3421 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3481 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3541 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3601 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3661 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3721 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3781 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3841 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3901 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3961 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4021 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4081 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4141 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4201 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4261 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4321 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4381 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4441 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4501 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4561 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4621 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4681 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4741 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4801 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4861 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacct
//
