LOCUS       sequence-482292-        8672 bp    DNA     circular SYN 06-AUG-2025
DEFINITION  Lentiviral expression of mutant Rac1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8672)
  AUTHORS   Weiss BG, Keth JM, Bhatt K, Doyal M, Hahn KM, Noh J, Isogai T, 
            Danuser G
  TITLE     Morphological control of merlin-Rac antagonism in 
            proliferation-promoting signaling.
  JOURNAL   Sci Signal. 2025 May 20;18(887):eadk0922. doi: 
            10.1126/scisignal.adk0922. Epub 2025 May 20.
  PUBMED    40392939
REFERENCE   2  (bases 1 to 8672)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci 
            Signal."; date: "2025-05-20"; volume: "18(887):eadk0922. doi"; 
            pages: " 10.1126/scisignal.adk0922. Epub 2025 May 20"
COMMENT     Sequence Label: pLVX-IRES-PURO-RAC1-R66A
FEATURES             Location/Qualifiers
     source          1..8672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     LTR             1119..1752
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    1959..2547
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             2065..2076
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2474..2494
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2561..3160)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2644..2664)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3141..3160
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     misc_feature    join(3180..3188,3189..3191,3192..3766)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3353..3372)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     3582..3599
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(3801..4379)
                     /codon_start=1
                     /product="human Rac family small GTPase 1"
                     /label=Rac1
                     /translation="MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGQEDYDALRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     primer_bind     complement(4459..4483)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(4480..4683)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4509..4529)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4684..4987
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    5045..5160
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     CDS             complement(5423..5467)
                     /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    5652..5885
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    6382..6507
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6554..7187
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     polyA_signal    7355..7489
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7379..7398)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7433..7452
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        7537..7641
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7642..7710,7711..8502)
                     /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(7860..7879)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtaaa cgctagcaat gtcaaggcct ctcactctct gatattcatt tctttgcaag
     1081 ttataaatac tgaataataa gatgacatga actactactg ctagagattt tccacactga
     1141 ctaaaagggt ctgagggatc tctagttacc agagtcacac aacagacggg cacacactac
     1201 ttgaagcact caaggcaagc tttattgagg cttaagcagt gggttcccta gttagccaga
     1261 gagctcccag gctcagatct ggtctaacca gagagaccca gtacaggcaa aaagcagctg
     1321 cttatatgca ggatctgagg gctcgccact ccccagtccc gcccaggcca cgcctccctg
     1381 gaaagtcccc agcggaaagt cccttgtagc aagctcgata tcagcagttc ttgaagtact
     1441 ccggatgcag ctctcgggcc acgtgatgaa atgctaggcg gctgtcaaac ctccactcta
     1501 acacttctct ctccgggtca tccatcccat gcaggctcac agggtgtaac aagctggtgt
     1561 tctctccttt attggcctct tctaccttat ctggctcaac tggtactagc ttgtagcacc
     1621 atccaaaggt cagtggatat ctgacccctg gccctggtgt gtagttctgc taatcaggga
     1681 agtagccttg tgtgtggtag atccacagat caaggatatc ttgtcttcgt tgggagtgaa
     1741 ttagcccttc cagtcccctc ttttctttta aaaagtggct aagatctaca gctgccttgt
     1801 aagtcattgg tcttaaaggt acctgaggtg tgactggaaa acccacctcc tcctcctctt
     1861 gtgcttctag ccaggcacaa tcagcattgg tagctgctgt attgctactt gtgattgctc
     1921 catgtttttc taggtctcga ctgcagaatt aattccaggc ggggaggcgg cccaaaggga
     1981 gatccgactc gtctgagggc gaaggcgaag acgcggaaga ggccgcagag ccggcagcag
     2041 gccgcgggaa ggaaggtccg ctggattgag ggccgaaggg acgtagcaga aggacgtccc
     2101 gcgcagaatc caggtggcaa cacaggcgag cagccatgga aaggacgtca gcttccccga
     2161 caacaccacg gaattgtcag tgcccaacag ccgagcccct gtccagcagc gggcaaggca
     2221 ggcggcgatg agttccgccg tggcaatagg gagggggaaa gcgaaagtcc cggaaaggag
     2281 ctgacaggtg gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca cagtgcacac
     2341 cacgccacgt tgcctgacaa cgggccacaa ctcctcataa agagacagca accaggattt
     2401 atacaaggag gagaaaatga aagccatacg ggaagcaata gcatgataca aaggcattaa
     2461 agcagcgtat ccacatagcg taaaaggagc aacatagtta agaataccag tcaatctttc
     2521 acaaattttg taatccagag gttgattgtt ccagacgcgt ctaggcaccg ggcttgcggg
     2581 tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga
     2641 gccgctcgta gaaggggagg ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg
     2701 cgcgctcggc cgcctccact ccggggagca cgacggcgct gcccagaccc ttgccctggt
     2761 ggtcgggcga gacgccgacg gtggccagga accacgcggg ctccttgggc cggtgcggcg
     2821 ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga accgctcaac tcggccatgc
     2881 gcgggccgat ctcggcgaac accgcccccg cttcgacgct ctccggcgtg gtccagaccg
     2941 ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga
     3001 ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg gtccgggtcg acggtgtggc
     3061 gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg tacggcccgg gggacgtcgt
     3121 cgcgggtggc gaggcgcacc gtgggcttgt actcggtcat ggaaggtcgt ctccttgtgg
     3181 gttgtggcaa gcttatcatc gtgtttttca aaggaaaacc acgtccccgt ggttcggggg
     3241 gcctagacgt ttttttaacc tcgactaaac acatgtaaag catgtgcacc gaggccccag
     3301 atcagatccc atacaatggg gtaccttctg ggcatccttc agccccttgt tgaatacgct
     3361 tgaggagagc catttgactc tttccacaac tatccaactc acaacgtggc actggggttg
     3421 tgccgccttt gcaggtgtat cttatacacg tggcttttgg ccgcagaggc acctgtcgcc
     3481 aggtgggggg ttccgctgcc tgcaaagggt cgctacagac gttgtttgtc ttcaagaagc
     3541 ttccagagga actgcttcct tcacgacatt caacagacct tgcattcctt tggcgagagg
     3601 ggaaagaccc ctaggaatgc tcgtcaagaa gacagggcca ggtttccggg ccctcacatt
     3661 gccaaaagac ggcaatatgg tggaaaataa catatagaca aacgcacacc ggccttattc
     3721 caagcggctt cggccagtaa cgttaggggg gggggaggga gaggggcggg atccgcggcc
     3781 gctctagaac tagtctcgag ttacaacagc aggcattttc tcttcctctt cttcacggga
     3841 ggcgggcaga ggactgctcg gatcgcttcg tcaaacactg tcttgaggcc tcgctgtgtg
     3901 agcgccgagc actccaggta ttttacagca ccaatctcct tagccatggc tagaccctgc
     3961 ggataggtga tgggagtcag cttcttctcc ttcagtttct cgatcgtgtc tttatcatcc
     4021 ctaagatcaa gtttagttcc cactaggatg atgggagtgt tgggacagtg gtgccgcacc
     4081 tcaggatacc actttgcacg gacattttca aatgatgcag gactcacaag ggaaaagcaa
     4141 attaagaaca catctgtttg cggataggat agggggcgta atgcgtcata atcttcttgt
     4201 ccagctgtat cccataagcc cagattcacc ggttttccat ctaccataac attggcagaa
     4261 taattgtcaa agacagtagg gatatattct ccaggaaatg cattggttgt gtaactgatc
     4321 agtaggcaag ttttacctac agctccgtct cccaccacca cacacttgat ggcctgcatg
     4381 aattcaccgg aaatagatcc tctagtagag tcggtgtctt ctatggaggt caaaacagcg
     4441 tggatggcgt ctccaggcga tctgacggtt cactaaacga gctctgctta tatagacctc
     4501 ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg
     4561 aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact
     4621 tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat
     4681 caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa
     4741 ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg
     4801 cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc
     4861 cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat
     4921 tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta
     4981 tgtaacgcgg aactcccaag cttatcgata aactggatct ctgctgtccc tgtaataaac
     5041 ccgaaaattt tgaatttttg taatttgttt ttgtaattct ttagtttgta tgtctgttgc
     5101 tattatgtct actattcttt cccctgcact gtacccccca atcccccctt ttcttttaaa
     5161 ggcgataccg tcgagatccg ttcactaatc gaatggatct gtctctgtct ctctctccac
     5221 cttcttcttc tattccttcg ggcctgtcgg gtcccctcgg ggttgggagg tgggtctgaa
     5281 acgataatgg tgaatatccc tgcctaactc tattcactat agaaagtaca gcaaaaacta
     5341 ttcttaaacc taccaagcct cctactatca ttatgaataa ttttatatac cacagccaat
     5401 ttgttatgtt aaaccaattc cacaaacttg cccatttatc taattccaat aattcttgtt
     5461 cattcttttc ttgctggttt tgcgattctt caattaagga gtgtattaag cttgtgtaat
     5521 tgttaatttc tctgtcccac tccatccagg tcgtgtgatt ccaaatctgt tccagagatt
     5581 tattactcca actagcattc caaggcacag cagtggtgca aatgagtttt ccagagcaac
     5641 cccaaatccc caggagctgt tgatccttta ggtatctttc cacagccagg attcttgcct
     5701 ggagctgctt gatgccccag actgtgagtt gcaacagatg ctgttgcgcc tcaatagccc
     5761 tcagcaaatt gttctgctgc tgcactatac cagacaataa ttgtctggcc tgtaccgtca
     5821 gcgtcattga cgctgcgccc atagtgcttc ctgctgctcc caagaaccca aggaacaaag
     5881 ctcctattcc cactgctctt ttttctctct gcaccactct tctctttgcc ttggtgggtg
     5941 ctactcctaa tggttcaatt tttactactt tatatttata taattcactt ctccaattgt
     6001 ccctcatatc tcctcctcca ggtctgaaga tcagcggccg gccgcttgct gtgcggtggt
     6061 cttacttttg ttttgctctt cctctatctt gtctaaagct tccttggtgt cttttatctc
     6121 tatcctttga tgcacacaat agagggttgc tactgtatta tataatgatc taagttcttc
     6181 tgatcctgtc tgaagggatg gttgtagctg tcccagtatt tgtctacagc cttctgatgt
     6241 ttctaacagg ccaggattaa ctgcgaatcg ttctagctcc ctgcttgccc atactatatg
     6301 ttttaattta tattttttct ttccccctgg ccttaaccga attttttccc atcgcgatct
     6361 aattctcccc cgcttaatac tgacgctctc gcacccatct ctctccttct agcctccgct
     6421 agtcaaaatt tttggcgtac tcaccagtcg ccgcccctcg cctcttgccg tgcgcgcttc
     6481 agcaagccga gtcctgcgtc gagagagctc ctctggtttc cctttcgctt tcaagtccct
     6541 gttcgggcgc cactgctaga gattttccac actgactaaa agggtctgag ggatctctag
     6601 ttaccagagt cacacaacag acgggcacac actacttgaa gcactcaagg caagctttat
     6661 tgaggcttaa gcagtgggtt ccctagttag ccagagagct cccaggctca gatctggtct
     6721 aaccagagag acccagtaca ggcaaaaagc agctgcttat atgcaggatc tgagggctcg
     6781 ccactcccca gtcccgccca ggccacgcct ccctggaaag tccccagcgg aaagtccctt
     6841 gtagcaagct cgatatcagc agttcttgaa gtactccgga tgcagctctc gggccacgtg
     6901 atgaaatgct aggcggctgt caaacctcca ctctaacact tctctctccg ggtcatccat
     6961 cccatgcagg ctcacagggt gtaacaagct ggtgttctct cctttattgg cctcttctac
     7021 cttatctggc tcaactggta ctagcttgta gcaccatcca aaggtcagtg gatatctgac
     7081 ccctggccct ggtgtgtagt tctgctaatc agggaagtag ccttgtgtgt ggtagatcca
     7141 cagatcaagg atatcttgtc ttctttggga gtgaattagc ccttccaact actaagtttg
     7201 tagtacatat ttaacaaata caatttcttt aaaatgaaaa taattcagag gaatcacagg
     7261 tttagagtaa atgaaaccac aggtaattgg cagtggtaat agggtatggg gtgggaagtt
     7321 tgggatgatt ttggttagct tgagttatcc agttgatcca gacatgataa gatacattga
     7381 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     7441 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg atctcccgat
     7501 ccgtcgacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     7561 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     7621 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     7681 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     7741 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     7801 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     7861 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     7921 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     7981 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     8041 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     8101 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     8161 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     8221 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     8281 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     8341 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     8401 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     8461 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     8521 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     8581 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     8641 tcagaccccg tagaaaagat caaaggatct tc
//
