LOCUS       sequence-414082-        8517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To reconstitute CD20 expression with a specific mRNA isoform 
            (variant 3) after CRISPR-Cas9-mediated knockout of CD20..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8517)
  AUTHORS   Ang Z, Paruzzo L, Hayer KE, Schmidt C, Torres-Diz M, Xu F, Zankharia
            U, Zhang Y, Soldan SS, Zheng S, Falkenstein CD, Loftus JP, Yang SY, 
            Asnani M, King Sainos P, Pillai V, Chong ER, Li M, Tasian SK, Barash
            Y, Lieberman PM, Ruella M, Schuster SJ, Thomas-Tikhonenko A
  TITLE     Alternative splicing of its 5' UTR limits CD20 mRNA translation and 
            enables resistance to CD20-directed immunotherapies.
  JOURNAL   Blood. 2023 Sep 8:blood.2023020400. doi: 10.1182/blood.2023020400.
  PUBMED    37683180
REFERENCE   2  (bases 1 to 8517)
  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: "Blood. 2023
            Sep 8:blood.2023020400. doi: 10.1182/blood.2023020400."
COMMENT     Sequence Label: pCDH-V3-rCD20-BSD
FEATURES             Location/Qualifiers
     source          1..8517
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1807..1923
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1966..2177
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2190..2458
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     misc_feature    3605..4174
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3624..4176
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3768..3785)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3995..4014
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4188..4583
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    4596..5184
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4649..4669)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5067..5078)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5258..5491
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5563..5684
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5600..5619)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5654..5673
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5724..5859
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5892..5908)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5892..5908)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5905..5927)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5916..5932
                     /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(5940..5946,5947..5964,5965..5970))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5985..6006
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6123..6140)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6294..6882)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(7053..7844,7845..7913))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGVGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7676..7695
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7914..8018)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8086..8104
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8142..8164)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8264..8283
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8477..8499
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8491..8508
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8492..8508
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccact gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     1741 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg
     1801 gttaactttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     1861 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aattcaaaat
     1921 tttatcgata ctagtggatc tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat
     1981 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt gcctagagaa
     2041 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     2101 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     2161 ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca cgcgcccgcc
     2221 gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg
     2281 gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg agaccgggcc
     2341 tttgtccggc gctcccttgg agcctaccta gactcagccg gctctccacg ctttgcctga
     2401 ccctgcttgc tcaactctac gtctttgttt cgttttctgt tctgcgccgt tacagatcca
     2461 agctgtgacc ggcgcctact ctagaggtct atcagcgatt tcatcttcag gcctggacta
     2521 caccactcac cctcccagtg tgcttgagaa acaaactgca cccactgaac tccgcagcta
     2581 gcatccaaat cagcccttga gatttgaggc cttggagact caggagtttt gagagcaaaa
     2641 tgacaacacc cagaaattca gtaaatggga ctttcccggc agagccaatg aaaggcccta
     2701 ttgctatgca atctggtcca aaaccactct tcaggaggat gtcttcactg gtgggcccca
     2761 cgcaaagctt cttcatgagg gaatctaaga ctttgggggc tgtccagatt atgaatgggc
     2821 tcttccacat tgcgttgggc ggactactta tgattccagc agggatctat gcacccatct
     2881 gtgtgactgt gtggtaccct ctctggggag gcattatgta tattatttcc ggatcactcc
     2941 tggcagcaac ggagaaaaac tccaggaagt gtttggtcaa aggaaaaatg ataatgaatt
     3001 cattgagcct ctttgctgcc atttctggaa tgattctttc aatcatggac atacttaata
     3061 ttaaaatttc ccatttttta aaaatggaga gtctgaattt tattagagct cacacaccat
     3121 atattaacat atacaactgt gaaccagcta atccctctga gaaaaactcc ccatctaccc
     3181 aatactgtta cagcatacaa tctctgttct tgggcatttt gtcagtgatg ctgatctttg
     3241 ccttcttcca ggaacttgta atagctggca tcgttgagaa tgaatggaaa agaacgtgct
     3301 ccagacccaa atctaacata gttctcctgt cagcagaaga aaaaaaagaa cagactattg
     3361 aaataaaaga agaagtggtt gggctaactg aaacatcttc ccaaccaaag aatgaagaag
     3421 acattgaaat tattccaatc caagaagagg aagaagaaga aacagagacg aactttccag
     3481 aacctcccca agatcaggaa tcctcaccaa tagaaaatga cagctctcct taaggatccc
     3541 agtgtggtgg tacgggaatt cctgcaggcc tcgagggccg gcgcgccgcg gccgctacgt
     3601 aaattccgcc cccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
     3661 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg
     3721 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
     3781 aatgcaaggt cttttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     3841 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     3901 ctgcgtccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     3961 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     4021 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
     4081 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     4141 ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg gttcctttgt
     4201 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
     4261 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
     4321 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
     4381 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
     4441 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag
     4501 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
     4561 ctggttatgt gtgggagggc taaatctagg tcgacaatca acctctggat tacaaaattt
     4621 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     4681 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     4741 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     4801 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     4861 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     4921 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     4981 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     5041 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     5101 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     5161 tctccctttg ggccgcctcc ccgcctggta cctttaagac caatgactta caaggcagct
     5221 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     5281 cgaagataag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     5341 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     5401 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     5461 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag
     5521 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     5581 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5641 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     5701 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     5761 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     5821 agctattcca gaagtagtga ggaggctttt ttggaggcct agacttttgc agagaccaaa
     5881 ttcgtaatca tgtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5941 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     6001 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     6061 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     6121 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     6181 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     6241 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6301 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6361 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6421 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6481 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6541 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6601 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6661 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6721 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6781 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6841 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6901 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6961 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     7021 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     7081 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     7141 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     7201 cgctcaccga ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7261 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7321 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7381 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7441 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7501 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7561 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7621 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7681 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7741 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7801 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7861 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7921 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7981 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     8041 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     8101 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     8161 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     8221 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     8281 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     8341 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     8401 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     8461 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagctg
//
