LOCUS       sequence-223049-        7305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of full length Rag2 with T490A mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7305)
  AUTHORS   Grundy GJ, Yang W, Gellert M
  TITLE     Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome
            by an epigenetic signal in V(D)J recombination.
  JOURNAL   Proc Natl Acad Sci U S A. 2010 Dec 28;107(52):22487-92. doi: 
            10.1073/pnas.1014958107. Epub 2010 Dec 13.
  PUBMED    21149691
REFERENCE   2  (bases 1 to 7305)
  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"; doi: 
            "10.1073/pnas.1014958107"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2010-12-28- 28"; volume: "107"; issue: "52"; pages: 
            "22487-92"
COMMENT     Sequence Label: Rag2 T490A
FEATURES             Location/Qualifiers
     source          1..7305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          88..1104
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1028..1050
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1112..1131
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1180..1189
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1192..1215
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1222..2319
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2176..2185
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2293..2316
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2368..2391
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(4084..4103)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4149..4204
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4203..4222)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4565..4581)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4565..4581)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4578..4600)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4589..4605
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4613..4619,4620..4637,4638..4643))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4658..4679
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4808..4825)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4979..5567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5059..5078)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5738..6529,6530..6598))
                     /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     6361..6380
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6599..6703)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6734..7113
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(7115..7305,1..87)
                     /label=chicken beta-actin promoter
ORIGIN
        1 aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc
       61 tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc
      121 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
      181 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg
      241 cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc
      301 ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc
      361 tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc
      421 agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag
      481 gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg
      541 tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct
      601 tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg
      661 cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag
      721 gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc
      781 cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag
      841 ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc
      901 gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt
      961 ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc
     1021 agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt
     1081 catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat
     1141 cattttggca aagaattgcg gccgtctcag gccgaattcg ccaccatggt gcatcaccat
     1201 caccatcacc atcatcgatc catgatcgaa gaaggtaaac tggtaatctg gattaacggc
     1261 gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt
     1321 aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact
     1381 ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct
     1441 ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc
     1501 tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta
     1561 tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg
     1621 gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa
     1681 ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac
     1741 ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc
     1801 ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca
     1861 gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc
     1921 aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa
     1981 ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa
     2041 gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt
     2101 aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa
     2161 gatccacgta ttgccgccac catggaaaac gcccagaaag gtgaaatcat gccgaacatc
     2221 ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt
     2281 cgtcagactg tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac
     2341 aacaataaca ataacaacaa cctcgggctc gaagtcctat tccagggacc ggtgtccctg
     2401 cagatggtaa cagtgggtca taacatagcc ttaattcaac caggcttctc acttatgaat
     2461 tttgatggcc aagttttctt ctttggccag aaaggctggc ctaagagatc ctgtcctact
     2521 ggagtctttc attttgatat aaaacaaaat catctcaaac tgaagcctgc aatcttctct
     2581 aaagattcct gctacctccc acctcttcgt tatccagcta cttgctcata caaaggcagc
     2641 atagactctg acaagcatca atatatcatt cacggaggga aaacaccaaa caatgagctt
     2701 tccgataaga tttatatcat gtctgtcgct tgcaagaata acaaaaaagt tactttccgt
     2761 tgcacagaga aagacttagt aggagatgtc cctgaaccca gatacggcca ttccattgac
     2821 gtggtgtata gtcgagggaa aagcatgggt gttctctttg gaggacgttc atacatgcct
     2881 tctacccaga gaaccacaga aaaatggaat agtgtagctg actgcctacc ccatgttttc
     2941 ttgatagatt ttgaatttgg gtgtgctaca tcatatattc tcccagaact tcaggatggg
     3001 ctgtcttttc atgtttctat tgccagaaac gataccgttt atattttggg aggacactca
     3061 cttgccagta atatacgccc tgctaacttg tatagaataa gagtggacct tcccctgggt
     3121 accccagcag tgaattgcac agtcttgcca ggaggaatct ctgtctccag tgcaatcctc
     3181 actcaaacaa acaatgatga atttgttatt gtgggtggtt atcagctgga aaatcagaaa
     3241 aggatggtct gcagccttgt ctctctaggg gacaacacga ttgaaatcag tgagatggag
     3301 actcctgact ggacctcaga tattaagcat agcaaaatat ggtttggaag caacatggga
     3361 aacgggacta ttttccttgg cataccagga gacaataagc aggctatgtc agaagcattc
     3421 tatttctata ctttgagatg ctctgaagag gatttgagtg aagatcagaa aattgtctcc
     3481 aacagtcaga catcaacaga agatcctggg gactccactc cctttgaaga ctcagaggaa
     3541 ttttgtttca gtgctgaagc aaccagtttt gatggtgacg atgaatttga cacctacaat
     3601 gaagatgatg aagatgacga gtctgtaacc ggctactgga taacatgttg ccctacttgt
     3661 gatgttgaca tcaatacctg ggttccgttc tattcaacgg agctcaataa acccgccatg
     3721 atctattgtt ctcatgggga tgggcactgg gtacatgccc agtgcatgga tttggaagaa
     3781 cgcacactca tccacttgtc agaaggaagc aacaagtatt attgcaatga acatgtacag
     3841 atagcaagag cattgcaagc tcccaaaaga aaccccccct tacaaaaacc tccaatgaaa
     3901 tccctccaca aaaaaggctc tgggaaagtc ttgactcctg ccaagaaatc cttccttaga
     3961 agactgtttg attaataata actcgagact agtatcgcga taattcactc ctcaggtgca
     4021 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     4081 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     4141 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     4201 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     4261 ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag
     4321 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
     4381 gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct
     4441 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
     4501 catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc ttggcgtaat
     4561 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4621 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4681 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatcgatc
     4741 cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4801 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4861 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4921 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4981 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5041 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5101 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5161 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5221 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5281 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5341 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5401 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5461 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5521 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5581 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5641 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5701 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5761 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5821 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5881 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5941 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6001 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6061 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6121 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6181 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6241 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6301 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6361 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6421 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6481 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6541 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6601 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6661 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6721 tgccacctgg gtcgacattg attattgact agttattaat agtaatcaat tacggggtca
     6781 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     6841 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     6901 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     6961 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     7021 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     7081 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
     7141 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
     7201 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg
     7261 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagcc
//
