LOCUS       sequence-244314-        5815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template to knock-in mouse IgG2b Fc domain in rat IgG2a heavy 
            chain locus. Use in combination with PX458-gR2A_ISO to convert rat 
            IgG2a hybridoma to mouse IgG2b producing cell lines..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5815)
  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 5815)
  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>mG2b-srt-his
FEATURES             Location/Qualifiers
     source          1..5815
                     /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             2073..2090
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    2107..2680
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2130..2682
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2274..2291)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2501..2520
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2697..2706
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2703..3101
                     /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(4021..4038)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4131..4719)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4211..4230)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4893..5687)
                     /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(5688..5792)
                     /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 aacaacaccc ccatcagtct atccactggc ccctgggtgt ggagatacaa
     1081 ctggttcctc tgtgactctg ggatgcctgg tcaagggcta cttccctgag tcagtgactg
     1141 tgacttggaa ctctggatcc ctgtccagca gtgtgcacac cttcccagct ctcctgcagt
     1201 ctggactcta cactatgagc agctcagtga ctgtcccctc cagcacctgg ccaagtcaga
     1261 ccgtcacctg cagcgttgct cacccagcca gcagcaccac ggtggacaaa aaacttgagc
     1321 ccagcgggcc catttcaaca atcaacccct gtcctccatg caaggagtgt cacaaatgcc
     1381 cagctcctaa cctggagggt ggaccatccg tcttcatctt ccctccaaat atcaaggatg
     1441 tactcatgat ctccctgaca cccaaggtca cgtgtgtggt ggtggatgtg agcgaggatg
     1501 acccagacgt ccggatcagc tggtttgtga acaacgtgga agtacacaca gctcagacac
     1561 aaacccatag agaggattac aacagtacta tccgggtggt cagtgccctc cccatccagc
     1621 accaggactg gatgagtggc aaggagttca aatgcaaggt caacaacaaa gacctcccat
     1681 cacccatcga gagaaccatc tcaaaaatta aagggctagt cagagctcca caagtataca
     1741 tcttgccgcc accagcagag cagttgtcca ggaaagatgt cagtctcact tgcctggtcg
     1801 tgggcttcaa ccctggagac atcagtgtgg agtggaccag caatgggcat actgaggaga
     1861 actacaagga caccgcacca gtcctggact ctgacggttc ttacttcata tacagcaagc
     1921 tcgatataaa aacaagcaag tgggagaaaa cagattcctt ctcatgcaac gtgagacacg
     1981 agggtctgaa aaattactac ctgaagaaga ccatctcccg gtctccgggt aaacgtacgg
     2041 gaggcggagg cagcctgccg gaaaccggcg gccatcatca tcatcatcat tgagtcgacg
     2101 tcgaggcccc tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg
     2161 ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag
     2221 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc
     2281 caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg
     2341 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag
     2401 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca
     2461 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt
     2521 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc
     2581 tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa
     2641 ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca gaattcgcca
     2701 ccatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa
     2761 tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg
     2821 gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg
     2881 tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg
     2941 gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc
     3001 tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga
     3061 ttcgtgaatt gctgccctct ggttatgtgt gggagggcta agtactagtc gagtgtgcct
     3121 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3181 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3241 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3301 aatagcaggc atgctgggga tgcggtgggc tctatggaga tcttgtacaa cttggggagg
     3361 gtacaaaatg gaggacttgt aggagcttgg gtccagacct gtcagacaaa atgatcacgc
     3421 atacttattc ttgttgaaac aacagcccca tctgtctatc cactggctcc tggaactgct
     3481 ctcaaaagta actccatggt gaccctggga tgcctggtca agggctattt ccctgagtca
     3541 gtcaccgtga cctggaactc tggagccctg tccagcggtg tgcacacctt cccagctgtc
     3601 ctgcagtctg gactctacac tctcaccagc tcagtgactg taccctccag cacctggtcc
     3661 agccaggccg tcacctgcaa cgtagcccac ccggccagca gcaccaaggt ggacaagaaa
     3721 attggtgaga gaacaaccag gggatgaggg gctcactaga ggtgaggata aggcattaga
     3781 ttgcctacac caaccagggt gggcagacat caccagggag ggggcctcag cccaggagac
     3841 caaaaattct cctttgtctc ccttctggag atttctatgt cctttacacc catttattaa
     3901 tattctctgg gcctcatggg ccttcctttc actgcccgct ttccagtcgg gaaacctgtc
     3961 gtgccagctg cattaacatg gtcatagctg tttccttgcg tattgggcgc tctccgcttc
     4021 ctcgctcact gactcgctgc gctcggtcgt tcgggtaaag cctggggtgc ctaatgagca
     4081 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4141 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4201 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4261 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4321 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4381 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4441 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4501 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4561 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4621 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4681 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4741 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4801 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4861 agtatatatg agtaaacttg gtctgacagt tattagaaaa attcatccag cagacgataa
     4921 aacgcaatac gctggctatc cggtgccgca atgccataca gcaccagaaa acgatccgcc
     4981 cattcgccgc ccagttcttc cgcaatatca cgggtggcca gcgcaatatc ctgataacga
     5041 tccgccacgc ccagacggcc gcaatcaata aagccgctaa aacggccatt ttccaccata
     5101 atgttcggca ggcacgcatc accatgggtc accaccagat cttcgccatc cggcatgctc
     5161 gctttcagac gcgcaaacag ctctgccggt gccaggccct gatgttcttc atccagatca
     5221 tcctgatcca ccaggcccgc ttccatacgg gtacgcgcac gttcaatacg atgtttcgcc
     5281 tgatgatcaa acggacaggt cgccgggtcc agggtatgca gacgacgcat ggcatccgcc
     5341 ataatgctca ctttttctgc cggcgccaga tggctagaca gcagatcctg acccggcact
     5401 tcgcccagca gcagccaatc acggcccgct tcggtcacca catccagcac cgccgcacac
     5461 ggaacaccgg tggtggccag ccagctcaga cgcgccgctt catcctgcag ctcgttcagc
     5521 gcaccgctca gatcggtttt cacaaacagc accggacgac cctgcgcgct cagacgaaac
     5581 accgccgcat cagagcagcc aatggtctgc tgcgcccaat catagccaaa cagacgttcc
     5641 acccacgctg ccgggctacc cgcatgcagg ccatcctgtt caatcatact cttccttttt
     5701 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5761 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac
//
