LOCUS       sequence-251832-        5797 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template to knock-in FC silent mIgG2a isotype in rat IgG2a heavy
            chain locus. Use in combination with PX458-gR2A_ISO to convert rat 
            IgG2a hybridoma to FC silent producing cell lines..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5797)
  AUTHORS   van der Schoot JMS, Fennemann FL, Valente M, Dolen Y, Hagemans IM, 
            Becker AMD, Le Gall CM, van Dalen D, Cevirgel A, van Bruggen JAC, 
            Engelfriet M, Caval T, Bentlage AEH, Fransen MF, Nederend M, Leusen 
            JHW, Heck AJR, Vidarsson G, Figdor CG, Verdoes M, Scheeren FA
  TITLE     Functional diversification of hybridoma-produced antibodies by 
            CRISPR/HDR genomic engineering.
  JOURNAL   Sci Adv. 2019 Aug 28;5(8):eaaw1822. doi: 10.1126/sciadv.aaw1822. 
            eCollection 2019 Aug.
  PUBMED    31489367
REFERENCE   2  (bases 1 to 5797)
  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: "Sci Adv."; 
            date: "2019-08-28"; volume: "5(8):eaaw1822. doi"; pages: " 
            10.1126/sciadv.aaw1822. eCollection 2019 Aug"
COMMENT     Sequence Label: pHybr_r2a>m2a(silent)-srt-his
FEATURES             Location/Qualifiers
     source          1..5797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     278..300
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     292..309
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             343..354
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2055..2072
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    2089..2662
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2112..2664
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2256..2273)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2483..2502
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2679..2688
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2685..3083
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(4003..4020)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4113..4701)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4193..4212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4875..5669)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(5670..5774)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca
      361 aggccgcata gaaagatctg agtagaacca aggtaaaaag tgtgggtaaa aacacatgtt
      421 cacaggcctg gctgacatga tgctgggcac gtatggaggc aaagtcaaga gggcagtgta
      481 agggccagaa gtgaatcctg acccaagaat agagagtgct aaacctacgt agatcgaagc
      541 caactaaaaa gacaagctac aaaacgaagc taaggccaga gatcttggac tgtgaagagt
      601 tcagagaacc taggatcagg aaccattagt aacaggccaa ggaagataga agctgcctag
      661 gacttggcaa gagccaacat ggttggactg gaaaagaaag gaggagacag aagacaggag
      721 agatgtgcca acttgatttt gggcttcact gttgtccata ctgtgtgcag ccatatggcc
      781 cacagataac aggtttagcc gaggaacaca gatacccaca ttggacaatg gtgggggaac
      841 acagataccc atactacagg gctctttagg gcatttcctg aaagtgtact aggagtggga
      901 ctgggctcaa agggattagg tgtgatctgg cctggtgagg ctgacattgg caagcccaat
      961 ggttgggtgt tgcctcctcc atgtgctagc gatcgcaggc gcaatcttcg catttctttt
     1021 ttccagctaa gacaacagcc ccatctgtct atccactggc tcctgtatgt ggtgacacaa
     1081 ctggctcctc ggtgaccctg ggatgcctgg tcaagggcta tttccctgag ccagtcacct
     1141 tgacctggaa ctctggatcc ctgtccagcg gtgtgcacac cttcccagct gtcctgcagt
     1201 ctgatctcta cactctcagt tcctcagtga ctgtaacttc cagcacctgg cccagccaga
     1261 gcataacctg caacgtagcc cacccggcca gcagcaccaa ggtggacaag aaaattgagc
     1321 caaggggacc cactattaag ccatgtccac catgcaaatg cccagcacca aatgccgccg
     1381 gtggtccttc tgtcttcatc ttccccccaa agatcaaaga tgtgctcatg atctctttga
     1441 gtcctattgt cacgtgtgtt gtggtagacg ttagcgagga tgatcccgac gtccagatca
     1501 gctggtttgt aaataacgtg gaagtccaca cagctcagac tcagacccac cgggaggact
     1561 acgccagcac tttaagggta gtctctgcac tccccatcca gcaccaggac tggatgtctg
     1621 gcaaggagtt caaatgcaaa gtcaacaaca aggatttacc tgcccccatc gagcgaacca
     1681 tctccaaacc caaaggcagt gtacgagccc cacaggtata cgtgctgccc cctcccgagg
     1741 aagagatgac caagaagcaa gtcactctca cctgcatggt aacagatttc atgcccgaag
     1801 acatttatgt ggagtggacg aacaacggga agaccgagct aaactacaag aacactgaac
     1861 ctgtgttgga ctcagatggg agttacttca tgtactccaa gctccgtgta gagaaaaaga
     1921 actgggttga gagaaacagc tactcgtgta gcgtggtgca tgagggcctg cacaaccacc
     1981 atactacgaa gtctttctcc cggacacctg gtaaacgtac gggaggcgga ggcagcctgc
     2041 cggaaaccgg cggccatcac catcatcacc attgagtcga cgtcgaggcc cctctccctc
     2101 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     2161 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     2221 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     2281 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     2341 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     2401 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     2461 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     2521 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     2581 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     2641 cctttgaaaa acacgatgat aatatggcca cagaattcgc caccatggcc aagcctttgt
     2701 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
     2761 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
     2821 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
     2881 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
     2941 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag
     3001 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
     3061 ctggttatgt gtgggagggc taagtactag tcgagtgtgc cttctagttg ccagccatct
     3121 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     3181 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     3241 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     3301 gatgcggtgg gctctatgga gatcttgtac aacttgggga gggtacaaaa tggaggactt
     3361 gtaggagctt gggtccagac ctgtcagaca aaatgatcac gcatacttat tcttgttgaa
     3421 acaacagccc catctgtcta tccactggct cctggaactg ctctcaaaag taactccatg
     3481 gtgaccctgg gatgcctggt caagggctat ttccctgagt cagtcaccgt gacctggaac
     3541 tctggagccc tgtccagcgg tgtgcacacc ttcccagctg tcctgcagtc tggactctac
     3601 actctcacca gctcagtgac tgtaccctcc agcacctggt ccagccaggc cgtcacctgc
     3661 aacgtagccc acccggccag cagcaccaag gtggacaaga aaattggtga gagaacaacc
     3721 aggggatgag gggctcacta gaggtgagga taaggcatta gattgcctac accaaccagg
     3781 gtgggcagac atcaccaggg agggggcctc agcccaggag accaaaaatt ctcctttgtc
     3841 tcccttctgg agatttctat gtcctttaca cccatttatt aatattctct gggcctcatg
     3901 ggccttcctt tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaaca
     3961 tggtcatagc tgtttccttg cgtattgggc gctctccgct tcctcgctca ctgactcgct
     4021 gcgctcggtc gttcgggtaa agcctggggt gcctaatgag caaaaggcca gcaaaaggcc
     4081 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4141 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4201 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4261 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4321 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4381 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4441 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4501 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4561 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4621 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4681 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4741 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4801 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4861 tggtctgaca gttattagaa aaattcatcc agcagacgat aaaacgcaat acgctggcta
     4921 tccggtgccg caatgccata cagcaccaga aaacgatccg cccattcgcc gcccagttct
     4981 tccgcaatat cacgggtggc cagcgcaata tcctgataac gatccgccac gcccagacgg
     5041 ccgcaatcaa taaagccgct aaaacggcca ttttccacca taatgttcgg caggcacgca
     5101 tcaccatggg tcaccaccag atcttcgcca tccggcatgc tcgctttcag acgcgcaaac
     5161 agctctgccg gtgccaggcc ctgatgttct tcatccagat catcctgatc caccaggccc
     5221 gcttccatac gggtacgcgc acgttcaata cgatgtttcg cctgatgatc aaacggacag
     5281 gtcgccgggt ccagggtatg cagacgacgc atggcatccg ccataatgct cactttttct
     5341 gccggcgcca gatggctaga cagcagatcc tgacccggca cttcgcccag cagcagccaa
     5401 tcacggcccg cttcggtcac cacatccagc accgccgcac acggaacacc ggtggtggcc
     5461 agccagctca gacgcgccgc ttcatcctgc agctcgttca gcgcaccgct cagatcggtt
     5521 ttcacaaaca gcaccggacg accctgcgcg ctcagacgaa acaccgccgc atcagagcag
     5581 ccaatggtct gctgcgccca atcatagcca aacagacgtt ccacccacgc tgccgggcta
     5641 cccgcatgca ggccatcctg ttcaatcata ctcttccttt ttcaatatta ttgaagcatt
     5701 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5761 ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
