LOCUS       sequence-459544-        8846 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of shRNA targeting Cavin1 + reconstitution of 
            zebrafish mutant Cavin1 (Del4UC1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8846)
  AUTHORS   Brooks JW, Tillu V, Eckert J, Verma S, Collins BM, Parton RG, Yap AS
  TITLE     Caveola mechanotransduction reinforces the cortical cytoskeleton to 
            promote epithelial resilience.
  JOURNAL   Mol Biol Cell. 2023 Nov 1;34(12):ar120. doi: 
            10.1091/mbc.E23-05-0163. Epub 2023 Sep 6.
  PUBMED    37672337
REFERENCE   2  (bases 1 to 8846)
  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: "Mol Biol 
            Cell."; date: "2023-11-1"; volume: "34(12):ar120. doi"; pages: " 
            10.1091/mbc.E23-05-0163. Epub 2023 Sep 6"
COMMENT     Sequence Label: pLL5.0_shRNAHsCavin1_Del4UC1_EGFP
FEATURES             Location/Qualifiers
     source          1..8846
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2616..2929
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2848..2868
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     3006..3022
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3064..3097)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3668..3677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4895..4904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4901..4903,4904..4906,4907..5620)
                     /codon_start=1
                     /product="blue variant of GFP"
                     /label=BFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4946..4967)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5207..5226)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5554..5575
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(5648..5681)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5737..6323
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5790..5810)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6208..6219)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6326..6342)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6852..7032
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7094..7682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7174..7193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7853..8644,8645..8713))
                     /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     8476..8495
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8714..8818)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgcgct ctagagatcc gacgccgcca tctctaggcc
     2641 cgcgccggcc ccctcgcaca gacttgtggg agaagctcgg ctactcccct gccccggtta
     2701 atttgcatat aatatttcct agtaactata gaggcttaat gtgcgataaa agacagataa
     2761 tctgttcttt ttaatactag ctacatttta catgataggc ttggatttct ataagagata
     2821 caaatactaa attattattt taaaaaacag cacaaaagga aactcaccct aactgtaaag
     2881 taattgtgtg ttttgagact ataaatatcc cttggagaaa agccttgttt caccttccac
     2941 gtcaagaaga tccgcgaggt tcaagagacc tcgcggatct tcttgacgtg gaaggtgttt
     3001 tttctcgagg tcgacggtat cgataagctc gcttcacgag attccagcag gtcgagggac
     3061 ctaataactt cgtatagcat acattatacg aagttatatt aagggttcca agcttaagcg
     3121 gccgctgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa
     3181 ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg aacagagaga
     3241 cagcagaata tgggccaaca aggatatctg tggtaagcag ttcctgcccc ggctcagggc
     3301 caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag aaccatcaga
     3361 tgtttccagg gtgccccaag gacctgaaaa tgaccctgtg ccttatttga actaaccaat
     3421 cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca
     3481 caacccctca ctcggcgcgc cagtcctccg atagactgcg tcgcccgggt acccgtattc
     3541 ccaataaagc ctcttgctgt ttgcatccga atcgtggact cgctgatcct tgggagggtc
     3601 tcctcagatt gattgactgc ccacctcggg ggtctttcat cgaattctgc agtcgacggt
     3661 accgcgggcc accatggagg tgattgaaga cagccgtctg catctggagg tgcccgagct
     3721 gcgcaagtct cttactgaga tctcagaaga tgaaatcaac ctcccggtct ctgatgatga
     3781 agacgaagcc accgccgaga acaaagctaa agatgctgcc gagaaagaag aggctgggga
     3841 gggtggcgag atgaatgggg tcatggtcct cacgctgctt gacaaaatca ttggagccgt
     3901 tgaccagatc cagcagacgc aggctgggtt ggaagcccgt cagcgggaca tggagagatc
     3961 tgtgacggga atccaggggg agctcaccaa actgtccaag agccacacca ccacagccaa
     4021 caccgtcaat aagatgctgg agaaggtgcg taaggtgagc gtgaacgtca agtcggtgcg
     4081 ggggaacctg gagaaacagg ctggccagat caaacgtctg gagagcaacg agaacgagct
     4141 cctaaagaga agaaacttta aagtcatgat ctatcaggat gaagtaaaag tgggttcgaa
     4201 agtgaatgtg gccaagtcta tgaagtatcc agagcctgtg gaaggggaag ctgagggtga
     4261 actcaaagct gcagcagaag aaggaggaga ggacaaagag aaagttaatc tggacctgtc
     4321 ctctgatgaa gaggaggtgg agattgagga gacaattgag gagtctcgag cagaacgcat
     4381 caagcggagc agcctgcaac gtgtccagaa catcaaaacc gttttctcca aggggaatat
     4441 ggagaaaacc aaacagaaga ccaaggagaa catagaaaag aaaatgggta aacttggcac
     4501 ccgcatgtcc gtgaaccccg agcgcaaaga gaagatgaag acttcacaga agaaggtgat
     4561 gaagtctttt actcctgacc acaccatcta cgcccgctcc aaaacggccg tctacaaggt
     4621 tccaccgttc acatttcatg tgaagaaaat tcgtgaagga gaagtggaga ttcagggaac
     4681 tgagatggtg gaagttgtcg gcgacacaga ggaggtggag aacgcagtga tggagggaga
     4741 ggaagatgca gagatggaga tggaagctgg agagatggtg gaggaagacg aggacgagac
     4801 agaagacagc cctgagatga gagcgctgct ggagaaagga gacgagctgg tgctggtgga
     4861 agatcatgac agagacagcg atgatccacc ggtcgccacc atggtgagca agggcgagga
     4921 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     4981 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     5041 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgagcca
     5101 cggcgtccag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     5161 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     5221 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     5281 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttcaa
     5341 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg cgaacttcaa
     5401 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     5461 ccccatcggc gacggccccg tgctgctgcc cgacagccac tacctgagca cccagtccgc
     5521 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     5581 cgccgggatc actctcggca tggacgagct gtacaagtaa cctgcaggca attcgtcgag
     5641 ggacctaata acttcgtata gcatacatta tacgaagtta tacatgttta agggttccgg
     5701 ttccactagg tacaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt
     5761 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     5821 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     5881 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     5941 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     6001 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     6061 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     6121 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
     6181 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgg
     6241 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     6301 ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac
     6361 atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag
     6421 cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga
     6481 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     6541 taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct
     6601 acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg
     6661 gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag
     6721 agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag
     6781 tattagagtg gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc
     6841 cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     6901 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     6961 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     7021 aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     7081 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     7141 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     7201 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     7261 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     7321 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     7381 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     7441 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     7501 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     7561 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     7621 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     7681 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     7741 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     7801 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     7861 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7921 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7981 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     8041 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     8101 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     8161 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     8221 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     8281 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     8341 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     8401 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     8461 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     8521 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     8581 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     8641 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     8701 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     8761 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     8821 catttccccg aaaagtgcca cctgac
//
