LOCUS       sequence-456435-        7928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knockout of MST1 and MST2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7928)
  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 7928)
  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: sgMST1/2-2
FEATURES             Location/Qualifiers
     source          1..7928
                     /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    2544..2661
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2730..2941
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2967..3761
                     /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(3021..3040)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3631..3650
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    3785..4373
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3838..3858)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4256..4267)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4445..4678
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4756..4877
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4793..4812)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4847..4866
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4917..5052
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5072..5091)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5073..5091)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5101..5118)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5101..5117)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5110..5132)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5259..5714
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5346..5365)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5556..5577
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5740..5844
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5845..5913,5914..6705)
                     /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(6063..6082)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6876..7464
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7365..7384
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7618..7635
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7752..7773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7788..7793,7794..7811,7812..7818)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7826..7842
                     /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     7831..7853
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7850..7866
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7850..7866
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7885..7905
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7887..7905
                     /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 acaccgatag gaacaccatt
     1981 ttggagtttc agagctatgc acaagagtgc atagcaagtt gaaataaggc tagtccgtta
     2041 tcaactttgc tggaaacagc aaagtggcac cgagtcggtg ctttttttgc gtgaccgagc
     2101 ttgtctgccc gcgcagtcca ctagacagac gcgcagaggt cgggcgggcc tagggccaat
     2161 ttcccatggt tcccttcatt tgcatatatg atgtaataag gttatggaga ctattaaact
     2221 tagccctaat caaactatat gatgataatg tgtgtggtac aaaaggtcat aacttattat
     2281 atactttgaa acttaaaaaa gggttacagt ttagtcacca taactgtaaa attttttcta
     2341 ttcttagctt tatatagttc ttgagaggcc atgtttatcg ttatggagta ctgtggcgcg
     2401 tttaagagct atgctggaaa cagcatagca agtttaaata aggctagtcc gttatcaact
     2461 ttgctggaaa cagcaaagtg gcaccgagtc ggtgcttttt ttgaattcta gatcttgaga
     2521 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
     2581 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
     2641 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggcgc
     2701 cggctcgagt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
     2761 gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa
     2821 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
     2881 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
     2941 ggtgtcgtga cgcgggatcc gccaccatga ttgaacaaga tggattgcac gcaggttctc
     3001 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3061 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3121 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     3181 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3241 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3301 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3361 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3421 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3481 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3541 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3601 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3661 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3721 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agtacagtag cgcgttaagt
     3781 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3841 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3901 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3961 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     4021 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     4081 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     4141 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     4201 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     4261 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     4321 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact
     4381 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     4441 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
     4501 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     4561 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     4621 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     4681 acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     4741 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4801 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4861 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     4921 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4981 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     5041 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     5101 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     5161 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     5221 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     5281 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5341 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5401 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5461 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5521 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5581 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5641 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5701 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5761 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5821 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5881 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5941 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6001 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6061 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6121 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6181 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6241 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6301 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6361 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6421 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6481 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6541 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6601 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6661 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6721 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6781 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6841 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6901 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6961 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7021 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7081 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7141 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7201 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7261 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7321 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7381 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7441 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7501 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7561 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     7621 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     7681 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     7741 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     7801 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     7861 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
     7921 ctgcaagc
//
