LOCUS       sequence-244320-        5845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template to knock-in mouse IgA Fc domain in rat IgG2a heavy 
            chain locus. Use in combination with PX458-gR2A_ISO to convert rat 
            IgG2a hybridoma to mouse IgA producing cell lines..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5845)
  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 5845)
  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>mA-srt-his
FEATURES             Location/Qualifiers
     source          1..5845
                     /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             2103..2120
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    2137..2710
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2160..2712
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2304..2321)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2531..2550
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2727..2736
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2733..3131
                     /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(4051..4068)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4161..4749)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4241..4260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4923..5717)
                     /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(5718..5822)
                     /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 ttccaggctc agagtctgcg agaaatccca ccatctaccc actgacactc ccaccagctc
     1081 tgtcaagtga cccagtgata atcggctgcc tgattcacga ttacttccct tccggcacga
     1141 tgaatgtgac ctggggaaag agtgggaagg atataaccac cgtaaacttc ccacctgccc
     1201 tggcctctgg gggacggtac accatgagca gccagttgac cctgccagct gtcgagtgcc
     1261 cagaaggaga atccgtgaaa tgttccgtgc aacatgactc taaccccgtc caagaattgg
     1321 atgtgaattg ctctggtcct actcctcctc ctcctattac tattccttcc tgccagccca
     1381 gcctgtcact gcagcggcca gctcttgagg acctgctcct gggttcagat gccagcatca
     1441 catgtactct gaatggcctg agaaatcctg agggagctgt cttcacctgg gagccctcca
     1501 ctgggaagga tgcagtgcag aagaaagctg tgcagaattc ctgcggctgc tacagtgtgt
     1561 ccagcgtcct gcctggctgt gctgagcgct ggaacagtgg cgcatcattc aagtgcacag
     1621 ttacccatcc tgagtctggc accttaactg gcacaattgc caaagtcaca gtgaacacct
     1681 tcccacccca ggtccacctg ctaccgccgc cgtcggagga gctggccctg aatgagctct
     1741 tgtccctgac atgcctggtg cgagctttca accctaaaga agtgctggtg cgatggctgc
     1801 atggaaatga ggagctgtcc ccagaaagct acctagtgtt tgagccccta aaggagccag
     1861 gcgagggagc caccacctac ctggtgacaa gcgtgttgcg tgtatcagct gaaacctgga
     1921 aacagggtga ccagtactcc tgcatggtgg gccacgaggc cttgcccatg aacttcaccc
     1981 agaagaccat cgaccgtctg tcgggtaaac ccaccaatgt cagcgtgtct gtgatcatgt
     2041 cagagggaga tggcatctgc taccgtacgg gaggcggagg cagcctgccg gaaaccggcg
     2101 gccatcatca tcatcatcat tgagtcgacg tcgaggcccc tctccctccc ccccccctaa
     2161 cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc
     2221 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac
     2281 gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt
     2341 gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg
     2401 caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata
     2461 agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga
     2521 aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt
     2581 accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc
     2641 gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac
     2701 acgatgataa tatggccaca gaattcgcca ccatggccaa gcctttgtct caagaagaat
     2761 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     2821 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     2881 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     2941 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     3001 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
     3061 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     3121 gggagggcta agtactagtc gagtgtgcct tctagttgcc agccatctgt tgtttgcccc
     3181 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3241 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3301 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3361 tctatggaga tcttgtacaa cttggggagg gtacaaaatg gaggacttgt aggagcttgg
     3421 gtccagacct gtcagacaaa atgatcacgc atacttattc ttgttgaaac aacagcccca
     3481 tctgtctatc cactggctcc tggaactgct ctcaaaagta actccatggt gaccctggga
     3541 tgcctggtca agggctattt ccctgagtca gtcaccgtga cctggaactc tggagccctg
     3601 tccagcggtg tgcacacctt cccagctgtc ctgcagtctg gactctacac tctcaccagc
     3661 tcagtgactg taccctccag cacctggtcc agccaggccg tcacctgcaa cgtagcccac
     3721 ccggccagca gcaccaaggt ggacaagaaa attggtgaga gaacaaccag gggatgaggg
     3781 gctcactaga ggtgaggata aggcattaga ttgcctacac caaccagggt gggcagacat
     3841 caccagggag ggggcctcag cccaggagac caaaaattct cctttgtctc ccttctggag
     3901 atttctatgt cctttacacc catttattaa tattctctgg gcctcatggg ccttcctttc
     3961 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaacatg gtcatagctg
     4021 tttccttgcg tattgggcgc tctccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4081 tcgggtaaag cctggggtgc ctaatgagca aaaggccagc aaaaggccag gaaccgtaaa
     4141 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4201 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4261 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4321 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4381 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4441 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4501 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4561 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4621 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4681 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4741 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4801 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4861 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4921 tattagaaaa attcatccag cagacgataa aacgcaatac gctggctatc cggtgccgca
     4981 atgccataca gcaccagaaa acgatccgcc cattcgccgc ccagttcttc cgcaatatca
     5041 cgggtggcca gcgcaatatc ctgataacga tccgccacgc ccagacggcc gcaatcaata
     5101 aagccgctaa aacggccatt ttccaccata atgttcggca ggcacgcatc accatgggtc
     5161 accaccagat cttcgccatc cggcatgctc gctttcagac gcgcaaacag ctctgccggt
     5221 gccaggccct gatgttcttc atccagatca tcctgatcca ccaggcccgc ttccatacgg
     5281 gtacgcgcac gttcaatacg atgtttcgcc tgatgatcaa acggacaggt cgccgggtcc
     5341 agggtatgca gacgacgcat ggcatccgcc ataatgctca ctttttctgc cggcgccaga
     5401 tggctagaca gcagatcctg acccggcact tcgcccagca gcagccaatc acggcccgct
     5461 tcggtcacca catccagcac cgccgcacac ggaacaccgg tggtggccag ccagctcaga
     5521 cgcgccgctt catcctgcag ctcgttcagc gcaccgctca gatcggtttt cacaaacagc
     5581 accggacgac cctgcgcgct cagacgaaac accgccgcat cagagcagcc aatggtctgc
     5641 tgcgcccaat catagccaaa cagacgttcc acccacgctg ccgggctacc cgcatgcagg
     5701 ccatcctgtt caatcatact cttccttttt caatattatt gaagcattta tcagggttat
     5761 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5821 cgcacatttc cccgaaaagt gccac
//
