LOCUS       sequence-457747-        7930 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knockout of NF2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7930)
  AUTHORS   Klingbeil O, Skopelitis D, Tonelli C, Yoshimoto T, Alpsoy A, 
            Panepinto MC, Minicozzi F, Merrill JR, Cafiero AM, Aggarwal D, Russo
            S, Ha T, Demerdash OE, Wee TL, Spector DL, Lyons SK, Tuveson DA, 
            Cifani P, Vakoc CR
  TITLE     MARK2/MARK3 kinases are catalytic co-dependencies of YAP/TAZ in 
            human cancer.
  JOURNAL   Cancer Discov. 2024 Jul 26. doi: 10.1158/2159-8290.CD-23-1529.
  PUBMED    39058094
REFERENCE   2  (bases 1 to 7930)
  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: "Cancer 
            Discov. 2024 Jul 26. doi: 10.1158/2159-8290.CD-23-1529."
COMMENT     Sequence Label: sgNF2
FEATURES             Location/Qualifiers
     source          1..7930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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"
     promoter        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2546..2663
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2732..2943
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2969..3763
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3023..3042)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3633..3652
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    3787..4375
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3840..3860)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4258..4269)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4447..4680
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4758..4879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4795..4814)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4849..4868
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4919..5054
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5074..5093)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5075..5093)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5103..5120)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5103..5119)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5112..5134)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5261..5716
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5348..5367)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5558..5579
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5742..5846
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5847..5915,5916..6707)
                     /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(6065..6084)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6878..7466
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7367..7386
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7620..7637
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7754..7775
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7790..7795,7796..7813,7814..7820)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7828..7844
                     /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)."
     primer_bind     7833..7855
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7852..7868
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7852..7868
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7887..7907
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7889..7907
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgcgag atggagttca
     1981 attgcggttt cagagctatg cacaagagtg catagcaagt tgaaataagg ctagtccgtt
     2041 atcaactttg ctggaaacag caaagtggca ccgagtcggt gctttttttg cgtgaccgag
     2101 cttgtctgcc cgcgcagtcc actagacaga cgcgcagagg tcgggcgggc ctagggccaa
     2161 tttcccatgg ttcccttcat ttgcatatat gatgtaataa ggttatggag actattaaac
     2221 ttagccctaa tcaaactata tgatgataat gtgtgtggta caaaaggtca taacttatta
     2281 tatactttga aacttaaaaa agggttacag tttagtcacc ataactgtaa aattttttct
     2341 attcttagct ttatatagtt cttgagaggc catgtttatc ggcttggtac gcagagcacc
     2401 ggtttaagag ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa
     2461 ctttgctgga aacagcaaag tggcaccgag tcggtgcttt ttttgaattc tagatcttga
     2521 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     2581 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     2641 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc
     2701 gccggctcga gtggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     2761 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta
     2821 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     2881 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     2941 caggtgtcgt gacgcgggat ccgccaccat gattgaacaa gatggattgc acgcaggttc
     3001 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     3061 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     3121 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     3181 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcag gaagggactg
     3241 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     3301 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     3361 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     3421 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     3481 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     3541 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     3601 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     3661 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     3721 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagtacagt agcgcgttaa
     3781 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     3841 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     3901 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     3961 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     4021 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     4081 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     4141 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     4201 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     4261 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     4321 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
     4381 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     4441 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
     4501 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     4561 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     4621 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     4681 gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     4741 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4801 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4861 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     4921 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4981 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     5041 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     5101 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     5161 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     5221 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     5281 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     5341 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5401 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5461 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5521 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5581 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     5641 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5701 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5761 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5821 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5881 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5941 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6001 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6061 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6121 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6181 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6241 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6301 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6361 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6421 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6481 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6541 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6601 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6661 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6721 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6781 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6841 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6901 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6961 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7021 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7081 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     7141 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     7201 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7261 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7321 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7381 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7441 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7501 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7561 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     7621 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     7681 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     7741 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     7801 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     7861 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
     7921 agctgcaagc
//
