LOCUS       sequence-482291-        8736 bp    DNA     circular SYN 06-AUG-2025
DEFINITION  Lentiviral expression of FLAG-tagged N-WASP WH1 domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8736)
  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 8736)
  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-NEO-N-WASP-WH1
FEATURES             Location/Qualifiers
     source          1..8736
                     /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..3355)
                     /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="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNILVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2672..2691)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3282..3301
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_feature    join(3356..3364,3365..3367,3368..3939)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3529..3548)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     3758..3775
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(4417..4440)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      4440..4449
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4523..4547)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(4544..4747)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4573..4593)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4748..5051
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    5109..5224
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     CDS             complement(5487..5531)
                     /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    5716..5949
                     /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    6446..6571
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6618..7251
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     polyA_signal    7419..7553
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7443..7462)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7497..7516
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        7601..7705
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7706..7774,7775..8566)
                     /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(7924..7943)
                     /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 tcagaagaac tcgtcaagaa
     2581 ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc
     2641 ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct
     2701 gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt
     2761 ccaccaagat attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg
     2821 gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt
     2881 ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat
     2941 gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg
     3001 catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc
     3061 ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag
     3121 ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt
     3181 cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca
     3241 gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata
     3301 gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca gccatggttg
     3361 tggcaagctt atcatcgtgt ttttcaaagg aaaaccacgt ccccgtggtt cggggggcct
     3421 agacgttttt ttaacctcga ctaaacacat gtaaagcatg tgcaccgagg ccccagatca
     3481 gatcccatac aatggggtac cttctgggca tccttcagcc ccttgttgaa tacgcttgag
     3541 gagagccatt tgactctttc cacaactatc caactcacaa cgtggcactg gggttgtgcc
     3601 gcctttgcag gtgtatctta tacacgtggc ttttggccgc agaggcacct gtcgccaggt
     3661 ggggggttcc gctgcctgca aagggtcgct acagacgttg tttgtcttca agaagcttcc
     3721 agaggaactg cttccttcac gacattcaac agaccttgca ttcctttggc gagaggggaa
     3781 agacccctag gaatgctcgt caagaagaca gggccaggtt tccgggccct cacattgcca
     3841 aaagacggca atatggtgga aaataacata tagacaaacg cacaccggcc ttattccaag
     3901 cggcttcggc cagtaacgtt agggggggag ggagaggggc gatcgacggc ccaaaaggtc
     3961 tgtaactgct tttcgaaatt tttttgcttc ttcttcattg gcaaaattaa gagcaacttg
     4021 acaagtatct ccagcaaagg tatgaaaata tcctctagga ctattatata caaagttatt
     4081 gtatagctct tgttcccaca atagtttccc atccttaatg tcaaatattc ttaaaaaata
     4141 agatctctgt ggattgtcct taacaagaca agcaacacca ctgcacttct ttgaccacat
     4201 acagttccga tctgctgcat ataactgcac cactgctgaa gacatagtca cacatttctt
     4261 gccgaggaaa gtgaagaggg actcgttctc ttgaggagtc aggagcaagg atcctacgtt
     4321 agtgactcgc cgaggtggag gaggttgctg ctgaacactg ctgcttccgc ttccgccgga
     4381 tgcgctgcct ccaccgctgc cagatctgag tccggacttg tcgtcatcgt ctttgtagtc
     4441 catggtggca ccggaaatag atcctctagt agagtcggtg tcttctatgg aggtcaaaac
     4501 agcgtggatg gcgtctccag gcgatctgac ggttcactaa acgagctctg cttatataga
     4561 cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt
     4621 ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga
     4681 gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac tgccaaaacc
     4741 gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc
     4801 ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag
     4861 ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat
     4921 actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca
     4981 ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa
     5041 gttatgtaac gcggaactcc caagcttatc gataaactgg atctctgctg tccctgtaat
     5101 aaacccgaaa attttgaatt tttgtaattt gtttttgtaa ttctttagtt tgtatgtctg
     5161 ttgctattat gtctactatt ctttcccctg cactgtaccc cccaatcccc ccttttcttt
     5221 taaaggcgat accgtcgaga tccgttcact aatcgaatgg atctgtctct gtctctctct
     5281 ccaccttctt cttctattcc ttcgggcctg tcgggtcccc tcggggttgg gaggtgggtc
     5341 tgaaacgata atggtgaata tccctgccta actctattca ctatagaaag tacagcaaaa
     5401 actattctta aacctaccaa gcctcctact atcattatga ataattttat ataccacagc
     5461 caatttgtta tgttaaacca attccacaaa cttgcccatt tatctaattc caataattct
     5521 tgttcattct tttcttgctg gttttgcgat tcttcaatta aggagtgtat taagcttgtg
     5581 taattgttaa tttctctgtc ccactccatc caggtcgtgt gattccaaat ctgttccaga
     5641 gatttattac tccaactagc attccaaggc acagcagtgg tgcaaatgag ttttccagag
     5701 caaccccaaa tccccaggag ctgttgatcc tttaggtatc tttccacagc caggattctt
     5761 gcctggagct gcttgatgcc ccagactgtg agttgcaaca gatgctgttg cgcctcaata
     5821 gccctcagca aattgttctg ctgctgcact ataccagaca ataattgtct ggcctgtacc
     5881 gtcagcgtca ttgacgctgc gcccatagtg cttcctgctg ctcccaagaa cccaaggaac
     5941 aaagctccta ttcccactgc tcttttttct ctctgcacca ctcttctctt tgccttggtg
     6001 ggtgctactc ctaatggttc aatttttact actttatatt tatataattc acttctccaa
     6061 ttgtccctca tatctcctcc tccaggtctg aagatcagcg gccggccgct tgctgtgcgg
     6121 tggtcttact tttgttttgc tcttcctcta tcttgtctaa agcttccttg gtgtctttta
     6181 tctctatcct ttgatgcaca caatagaggg ttgctactgt attatataat gatctaagtt
     6241 cttctgatcc tgtctgaagg gatggttgta gctgtcccag tatttgtcta cagccttctg
     6301 atgtttctaa caggccagga ttaactgcga atcgttctag ctccctgctt gcccatacta
     6361 tatgttttaa tttatatttt ttctttcccc ctggccttaa ccgaattttt tcccatcgcg
     6421 atctaattct cccccgctta atactgacgc tctcgcaccc atctctctcc ttctagcctc
     6481 cgctagtcaa aatttttggc gtactcacca gtcgccgccc ctcgcctctt gccgtgcgcg
     6541 cttcagcaag ccgagtcctg cgtcgagaga gctcctctgg tttccctttc gctttcaagt
     6601 ccctgttcgg gcgccactgc tagagatttt ccacactgac taaaagggtc tgagggatct
     6661 ctagttacca gagtcacaca acagacgggc acacactact tgaagcactc aaggcaagct
     6721 ttattgaggc ttaagcagtg ggttccctag ttagccagag agctcccagg ctcagatctg
     6781 gtctaaccag agagacccag tacaggcaaa aagcagctgc ttatatgcag gatctgaggg
     6841 ctcgccactc cccagtcccg cccaggccac gcctccctgg aaagtcccca gcggaaagtc
     6901 ccttgtagca agctcgatat cagcagttct tgaagtactc cggatgcagc tctcgggcca
     6961 cgtgatgaaa tgctaggcgg ctgtcaaacc tccactctaa cacttctctc tccgggtcat
     7021 ccatcccatg caggctcaca gggtgtaaca agctggtgtt ctctccttta ttggcctctt
     7081 ctaccttatc tggctcaact ggtactagct tgtagcacca tccaaaggtc agtggatatc
     7141 tgacccctgg ccctggtgtg tagttctgct aatcagggaa gtagccttgt gtgtggtaga
     7201 tccacagatc aaggatatct tgtcttcttt gggagtgaat tagcccttcc aactactaag
     7261 tttgtagtac atatttaaca aatacaattt ctttaaaatg aaaataattc agaggaatca
     7321 caggtttaga gtaaatgaaa ccacaggtaa ttggcagtgg taatagggta tggggtggga
     7381 agtttgggat gattttggtt agcttgagtt atccagttga tccagacatg ataagataca
     7441 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     7501 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttgatctcc
     7561 cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     7621 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     7681 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     7741 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     7801 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     7861 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     7921 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     7981 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     8041 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     8101 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     8161 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     8221 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     8281 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     8341 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     8401 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     8461 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     8521 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     8581 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     8641 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     8701 agcgtcagac cccgtagaaa agatcaaagg atcttc
//
