LOCUS       sequence-244317-        5779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template to knock-in mouse IgG1 Fc domain in rat IgG2a heavy 
            chain locus. Use in combination with PX458-gR2A_ISO to convert rat 
            IgG2a hybridoma to mouse IgG1 producing cell lines..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5779)
  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 5779)
  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>mG1-srt-his
FEATURES             Location/Qualifiers
     source          1..5779
                     /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             2037..2054
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    2071..2644
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2094..2646
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2238..2255)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2465..2484
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2661..2670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2667..3065
                     /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(3985..4002)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4095..4683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4175..4194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4857..5651)
                     /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(5652..5756)
                     /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 ttccagcaaa gaccacacca ccttctgtgt acccactcgc accaggcagc gccgctcaaa
     1081 ccaacagtat ggtgaccttg gggtgtcttg tgaagggcta ctttcccgag cccgttaccg
     1141 tcacctggaa ctccgggtct ctctcaagcg gcgttcacac cttccccgcc gtactgcaat
     1201 cagacctcta taccctgtct tccagcgtca ctgtacccag ttccacctgg cccagtgaaa
     1261 ctgtcacatg taatgtcgca cacccagctt cctccaccaa agtggacaaa aagatcgtgc
     1321 ctagggactg tggatgcaag ccatgtatct gcactgtccc cgaggtttca agtgtattta
     1381 tcttcccccc aaagcccaaa gatgtcctca ccataacact cacacccaag gtgacctgcg
     1441 tggtggtcga tatcagtaaa gatgaccccg aggtccaatt cagctggttt gtggatgatg
     1501 tagaggtcca cactgctcag actcagccac gggaagagca gtttaactca acatttcgga
     1561 gtgtgtccga actgccaatc atgcaccaag attggctcaa tgggaaagaa ttcaaatgca
     1621 gggtgaatag tgccgccttt cctgcaccaa tagagaaaac catatcaaag acaaagggca
     1681 ggcccaaggc ccctcaagtc tatactatac ctccacccaa ggagcaaatg gctaaagata
     1741 aggtaagcct cacctgcatg atcacagatt ttttcccaga agacataact gtcgaatggc
     1801 aatggaacgg ccaacctgca gaaaattaca agaatactca gcctattatg gacaccgatg
     1861 gcagctactt cgtgtattca aaactgaacg tacagaaatc taattgggaa gcagggaata
     1921 cattcacatg ctctgtgttg catgaagggc tccataacca ccacactgaa aagagcttga
     1981 gccactctcc cggaaagcgt acgggaggcg gaggcagcct gccggaaacc ggcggccatc
     2041 atcatcatca tcattgagtc gacgtcgagg cccctctccc tccccccccc ctaacgttac
     2101 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     2161 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     2221 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     2281 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     2341 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     2401 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     2461 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     2521 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     2581 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     2641 ataatatggc cacagaattc gccaccatgg ccaagccttt gtctcaagaa gaatccaccc
     2701 tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg
     2761 ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta
     2821 ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca
     2881 acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac
     2941 ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg
     3001 atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg
     3061 gctaagtact agtcgagtgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3121 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3181 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3241 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3301 gagatcttgt acaacttggg gagggtacaa aatggaggac ttgtaggagc ttgggtccag
     3361 acctgtcaga caaaatgatc acgcatactt attcttgttg aaacaacagc cccatctgtc
     3421 tatccactgg ctcctggaac tgctctcaaa agtaactcca tggtgaccct gggatgcctg
     3481 gtcaagggct atttccctga gtcagtcacc gtgacctgga actctggagc cctgtccagc
     3541 ggtgtgcaca ccttcccagc tgtcctgcag tctggactct acactctcac cagctcagtg
     3601 actgtaccct ccagcacctg gtccagccag gccgtcacct gcaacgtagc ccacccggcc
     3661 agcagcacca aggtggacaa gaaaattggt gagagaacaa ccaggggatg aggggctcac
     3721 tagaggtgag gataaggcat tagattgcct acaccaacca gggtgggcag acatcaccag
     3781 ggagggggcc tcagcccagg agaccaaaaa ttctcctttg tctcccttct ggagatttct
     3841 atgtccttta cacccattta ttaatattct ctgggcctca tgggccttcc tttcactgcc
     3901 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct
     3961 tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt
     4021 aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4081 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4141 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4201 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4261 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4321 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4381 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4441 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4501 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4561 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4621 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4681 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4741 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4801 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttattag
     4861 aaaaattcat ccagcagacg ataaaacgca atacgctggc tatccggtgc cgcaatgcca
     4921 tacagcacca gaaaacgatc cgcccattcg ccgcccagtt cttccgcaat atcacgggtg
     4981 gccagcgcaa tatcctgata acgatccgcc acgcccagac ggccgcaatc aataaagccg
     5041 ctaaaacggc cattttccac cataatgttc ggcaggcacg catcaccatg ggtcaccacc
     5101 agatcttcgc catccggcat gctcgctttc agacgcgcaa acagctctgc cggtgccagg
     5161 ccctgatgtt cttcatccag atcatcctga tccaccaggc ccgcttccat acgggtacgc
     5221 gcacgttcaa tacgatgttt cgcctgatga tcaaacggac aggtcgccgg gtccagggta
     5281 tgcagacgac gcatggcatc cgccataatg ctcacttttt ctgccggcgc cagatggcta
     5341 gacagcagat cctgacccgg cacttcgccc agcagcagcc aatcacggcc cgcttcggtc
     5401 accacatcca gcaccgccgc acacggaaca ccggtggtgg ccagccagct cagacgcgcc
     5461 gcttcatcct gcagctcgtt cagcgcaccg ctcagatcgg ttttcacaaa cagcaccgga
     5521 cgaccctgcg cgctcagacg aaacaccgcc gcatcagagc agccaatggt ctgctgcgcc
     5581 caatcatagc caaacagacg ttccacccac gctgccgggc tacccgcatg caggccatcc
     5641 tgttcaatca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5701 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5761 tttccccgaa aagtgccac
//
