LOCUS       sequence-243698-        6392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template to knock-in sortag and histag in rat IgG2a heavy chain 
            locus. Use in combination with PX459-gR2A_Hinge to convert rat IgG2a
            hybridoma to Fab' fragment producing cell lines..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6392)
  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 6392)
  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>Fab-srt-his
FEATURES             Location/Qualifiers
     source          1..6392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     57..79
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     71..88
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     72..88
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     95..114
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        95..113
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             796..813
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    836..1409
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    859..1411
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1003..1020)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1230..1249
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      1426..1435
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1432..1830
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    1842..2066
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(2618..2636)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2657..2673)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2657..2673)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2670..2692)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2681..2697
                     /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(2705..2711,2712..2729,2730..2735))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2750..2771
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2888..2905)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3059..3647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3139..3158)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(3771..3839,3840..4631)
                     /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     complement(3989..4008)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(4881..5675)
                     /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"
     CDS             complement(4881..5675)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4992..5011)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5602..5621
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6024..6326)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(6126..6145)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
ORIGIN
        1 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
       61 gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt
      121 gaatttagcg gccgccagtg tgatggatat ctgcagaatt cgccctttga ttacgccaag
      181 cttggtaccg agctcggatc ccactagtaa cggccgccag tgtgctggaa ttcgccctcc
      241 tggaactctg gagccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgga
      301 ctctacactc tcaccagctc agtgactgta ccctccagca cctggtccag ccaggccgtc
      361 acctgcaacg tagcccaccc ggccagcagc accaaggtgg acaagaaaat tggtgagaga
      421 acaaccaggg gatgaggggc tcactagagg tgaggataag gcattagatt gcctacacca
      481 accagggtgg gcagacatca ccagggaggg ggcctcagcc caggagacca aaaattctcc
      541 tttgtctccc ttctggagat ttctatgtcc tttacaccca tttattaata ttctgggtaa
      601 gatgcccttg catcatgaca tacagaggca gactagagta tcaacctgca aaaggtcata
      661 cccaggaaga gcctgccatg atcccacacc agaaccaacc tggggccttc tcacctatag
      721 accatactaa cacacagcct tctctctgca gtgccaaggg aaggaggcgg aggcagcctg
      781 ccggaaaccg gcggccacca tcaccatcac cattgaggat cccaattgct cgaggcccct
      841 ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
      901 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac
      961 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
     1021 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa
     1081 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
     1141 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg
     1201 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
     1261 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat
     1321 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg
     1381 tggttttcct ttgaaaaaca cgatgataat atggccacag aattcgccac catggccaag
     1441 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
     1501 cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
     1561 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
     1621 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
     1681 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
     1741 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
     1801 ctgccctctg gttatgtgtg ggagggctaa gtactagtcg actgtgcctt ctagttgcca
     1861 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     1921 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     1981 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     2041 tgctggggat gcggtgggct ctatggagat cttgtacagg taagtcacta ggactattac
     2101 tccagcccca gattcaaaaa atatcctcag aggcccatgt tagaggatga cacagctatt
     2161 gacctatttc tacctttctt cttcatctac aggctcagaa gtatcatctg tcttcatctt
     2221 ccccccaaag accaaagatg tgctcaccat cactctgact cctaaggtca cgtgtgttgt
     2281 ggtagacatt agccagaatg atcccgaggt ccggttcagc tggtttatag atgacgtgga
     2341 agtccacaca gctcagactc atgccccgga gaagcagtcc aacagcactt tacgctcagt
     2401 cagtgaactc cccatcgtgc accgggactg gctcaatggc aagacgttca aatgcaaagt
     2461 caacagtgga gcattccctg cccccatcga gaaaagcatc tccaaacccg aaggtgggag
     2521 cagcagggtg tgtggtgtag aagctgcagt aggccataga cagagcttga cttaactaga
     2581 cttaagggcg aattcgttta aacctgcagg actagtccct ttagtgaggg ttaattctga
     2641 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2701 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2761 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2821 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2881 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2941 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3001 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3061 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3121 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3181 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3241 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3301 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3361 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3421 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3481 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3541 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3601 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3661 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3721 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3781 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3841 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3901 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3961 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4021 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4081 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4141 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4201 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4261 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4321 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4381 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4441 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4501 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4561 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4621 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4681 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg agcggataca
     4741 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4801 tgccacctga tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     4861 aaattgtaag cgttaataat tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
     4921 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
     4981 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
     5041 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
     5101 caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgctcgc cttgagcctg
     5161 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
     5221 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
     5281 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
     5341 ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc
     5401 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
     5461 gtggccagcc acgatagccg cgctgcctcg tcttgcagtt cattcagggc accggacagg
     5521 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
     5581 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
     5641 ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct
     5701 tgatcagagc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt
     5761 actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt
     5821 gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc
     5881 tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg
     5941 ggtcagcacc gtttctgcgg actggctttc tacgtgaaaa ggatctaggt gaagatcctt
     6001 tttgataatc tcatgcctga catttatatt ccccagaaca tcaggttaat ggcgtttttg
     6061 atgtcatttt cgcggtggct gagatcagcc acttcttccc cgataacgga gaccggcaca
     6121 ctggccatat cggtggtcat catgcgccag ctttcatccc cgatatgcac caccgggtaa
     6181 agttcacggg agactttatc tgacagcaga cgtgcactgg ccagggggat caccatccgt
     6241 cgccccggcg tgtcaataat atcactctgt acatccacaa acagacgata acggctctct
     6301 cttttatagg tgtaaacctt aaactgccgt acgtataggc tgcgcaactg ttgggaaggg
     6361 cgatcggtgc gggcctcttc gctattacgc ca
//
