LOCUS       sequence-377692-        7914 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a sgRNA for 5' tagging to LMNA and contains a cassette 
            with eGFP flanked by homology arms for LMNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7914)
  AUTHORS   Thomsen EA, Skipper KA, Andersen S, Haslund D, Skov TW, Mikkelsen JG
  TITLE     CRISPR-Cas9-directed gene tagging using a single integrase-defective
            lentiviral vector carrying a transposase-based Cas9 off switch.
  JOURNAL   Mol Ther Nucleic Acids. 2022 Aug 4;29:563-576. doi: 
            10.1016/j.omtn.2022.08.005. eCollection 2022 Sep 13.
  PUBMED    36090759
REFERENCE   2  (bases 1 to 7914)
  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 Ther 
            Nucleic Acids."; date: "2022-08-4"; volume: "29:563-576. doi"; 
            pages: " 10.1016/j.omtn.2022.08.005. eCollection 2022 Sep 13"
COMMENT     Sequence Label: pLV.5'-LMNA-eGFP-KI
FEATURES             Location/Qualifiers
     source          1..7914
                     /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_RNA        1987..2062
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2118..2235
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             join(2664..2666,2667..2669,2670..3380)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2664..2666,2667..2669,2670..3380)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2709..2730)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2970..2989)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3317..3338
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3771..4359
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3824..3844)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4242..4253)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4431..4664
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4742..4863
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4779..4798)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4833..4852
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4903..5038
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5058..5077)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5059..5077)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5087..5104)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5087..5103)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5096..5118)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5245..5700
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5332..5351)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5542..5563
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5726..5830
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5831..5899,5900..6691)
                     /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(6049..6068)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6862..7450
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7351..7370
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7604..7621
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7738..7759
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7774..7779,7780..7797,7798..7804)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7812..7828
                     /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     7817..7839
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7836..7852
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7836..7852
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7871..7891
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7873..7891
                     /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 acaccgccat ggagaccccg
     1981 tcccaggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     2041 aaaagtggca ccgagtcggt gcttttttaa gcttggcgta actagatctt gagacaaatg
     2101 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2161 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2221 aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc
     2281 gagggggcca atgcggggcc cgactccgcc acaccccaac ggtccttccc cctcctcacc
     2341 actcccgccc ccacccccaa tggatctggg actgcccctt taagagtagt ggcccctcct
     2401 cccttcagag gaggacctat tagagccttt gccccggcgt cggtgactca gtgttcgcgg
     2461 gagcgccgca cctacaccag ccaacccaga tcccgaggtc cgacagcgcc cggcccagat
     2521 ccccacgcct gccaggagca agccgagagc cagccggccg gcgcactccg actccgagca
     2581 gtctctgtcc ttcgacccga gccccgcgcc ctttccggga cccctgcccc gcgggcagcg
     2641 ctgccaacct gccggccatg gagatggtga gcaagggcga ggagctgttc accggggtgg
     2701 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2761 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2821 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     2881 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2941 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     3001 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     3061 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     3121 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     3181 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     3241 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     3301 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     3361 gcatggacga gctgtacaag atcagatctc gagccaccac cccgtctcag cggcgcgcca
     3421 cccgcagcgg ggcgcaggcc agctccactc cgctgtcgcc cacccgcatc acccggctgc
     3481 aggagaagga ggacctgcag gagctcaatg atcgcttggc ggtctacatc gaccgtgtgc
     3541 gctcgctgga aacggagaac gcagggctgc gccttcgcat caccgagtct gaagaggtgg
     3601 tcagccgcga ggtgtccggc atcaaggccg cctacgaggc cgagctcggg gatgcccgca
     3661 agacccttga ctcagtagcc aaggagcgcg cccgcctgca gctggagctg agcaaagtgc
     3721 gtgaggagtt taaggagctg aaagcgcggt gagttcgccc ttaagtcgac aatcaacctc
     3781 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     3841 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     3901 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     3961 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     4021 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     4081 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     4141 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     4201 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     4261 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     4321 ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac
     4381 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     4441 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
     4501 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     4561 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     4621 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc
     4681 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     4741 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4801 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4861 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg
     4921 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4981 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     5041 gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     5101 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     5161 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     5221 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5281 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5341 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5401 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5461 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5521 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5581 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5641 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5701 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5761 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5821 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5881 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5941 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     6001 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     6061 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6121 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     6181 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6241 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6301 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6361 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6421 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6481 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6541 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6601 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6661 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6721 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6781 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6841 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6901 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6961 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7021 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7081 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7141 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7201 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7261 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7321 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7381 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7441 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7501 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     7561 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     7621 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     7681 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     7741 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     7801 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     7861 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagc
//
