LOCUS       sequence-377631-        7186 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses a sgRNA for 5' tagging to LMNA and contains a cassette 
            with eGFP without homology arms.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7186)
  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 7186)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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'eGFP/sgLMNA
FEATURES             Location/Qualifiers
     source          1..7186
                     /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(2296..2298,2299..2301,2302..3012)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2296..2298,2299..2301,2302..3012)
                     /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(2341..2362)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2602..2621)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2949..2970
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3043..3631
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3096..3116)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3514..3525)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3703..3936
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4014..4135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4051..4070)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4105..4124
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4175..4310
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4330..4349)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4331..4349)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4359..4376)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4359..4375)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4368..4390)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4517..4972
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4604..4623)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4814..4835
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4998..5102
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5103..5171,5172..5963)
                     /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(5321..5340)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6134..6722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6623..6642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6876..6893
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7010..7031
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7046..7051,7052..7069,7070..7076)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7084..7100
                     /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     7089..7111
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7108..7124
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7108..7124
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7143..7163
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7145..7163
                     /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 atgcgatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     2341 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     2401 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     2461 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     2521 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     2581 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     2641 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2701 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     2761 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     2821 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2881 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     2941 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     3001 gagctgtaca agatcagatc tcgagccacc ggttaagtcg acaatcaacc tctggattac
     3061 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3121 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     3181 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3241 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3301 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3361 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3421 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     3481 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3541 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3601 agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg
     3661 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     3721 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     3781 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     3841 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     3901 tcagaccctt ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc
     3961 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     4021 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4081 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4141 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4201 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4261 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     4321 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     4381 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4441 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4501 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4561 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4621 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4681 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     4741 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4801 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     4861 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4921 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     4981 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5041 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5101 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5161 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5221 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5281 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5341 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5401 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5461 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5521 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5581 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5641 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5701 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5761 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     5821 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5881 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5941 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6001 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6061 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6121 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6181 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6241 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6301 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6361 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6421 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6481 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6541 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6601 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6661 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6721 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6781 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6841 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6901 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6961 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     7021 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     7081 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     7141 gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagc
//
