LOCUS       sequence-329210-        7607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses S. pyogenes CRISPR chimeric RNA element with customizable 
            sgRNA from U6 promoter and GFP from SFFV promoter as the marker for 
            infection. 3rd generation lentiviral backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7607)
  TITLE     Ebert Lab plasmids
REFERENCE   2  (bases 1 to 7607)
  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"
COMMENT     Sequence Label: sgRNA.SFFV.GFP
FEATURES             Location/Qualifiers
     source          1..7607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /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        1718..1966
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1718..1738
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1889..1908
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1985..2060
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2108..2225
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2283..2689
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2724..2733
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2730..2732,2733..2735,2736..3449)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2730..2732,2733..2735,2736..3446)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2775..2796)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3036..3055)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3383..3404
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3465..4053
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3518..3538)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3936..3947)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4125..4358
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4436..4557
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4473..4492)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4527..4546
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4597..4732
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4752..4771)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4753..4771)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4781..4798)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4781..4797)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4790..4812)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4939..5394
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5026..5045)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5236..5257
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5420..5524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5525..5593,5594..6385)
                     /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(5743..5762)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6556..7144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7045..7064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7298..7315
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7432..7453
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7468..7473,7474..7491,7492..7498)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7506..7522
                     /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     7511..7533
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7530..7546
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7530..7546
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7565..7585
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7567..7585
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc
     1741 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
     1801 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     1861 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     1921 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggag acgtgaccgt
     1981 ctctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     2041 aagtggcacc gagtcggtgc ttttttgaat tctagatctt gagacaaatg gcagtattca
     2101 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     2161 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     2221 attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc gagctagctg
     2281 cagtaacgcc attttgcaag gcatggaaaa ataccaaacc aagaatagag aagttcagat
     2341 caagggcggg tacatgaaaa tagctaacgt tgggccaaac aggatatctg cggtgagcag
     2401 tttcggcccc ggcccggggc caagaacaga tggtcaccgc agtttcggcc ccggcccgag
     2461 gccaagaaca gatggtcccc agatatggcc caaccctcag cagtttctta agacccatca
     2521 gatgtttcca ggctccccca aggacctgaa atgaccctgc gccttatttg aattaaccaa
     2581 tcagcctgct tctcgcttct gttcgcgcgc ttctgcttcc cgagctctat aaaagagctc
     2641 acaacccctc actcggcgcg ccagtcctcc gacagactga gtcggccggt cgaatcaagc
     2701 ttatcgatac cgtcgacgga tccgccacca tggtgagcaa gggcgaggag ctgttcaccg
     2761 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     2821 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     2881 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     2941 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     3001 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     3061 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     3121 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     3181 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     3241 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     3301 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     3361 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     3421 ctctcggcat ggacgagctg tacaagtaaa cgcgttaagt cgacaatcaa cctctggatt
     3481 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3541 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3601 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     3661 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     3721 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     3781 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3841 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     3901 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3961 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     4021 cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa
     4081 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     4141 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     4201 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     4261 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     4321 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca
     4381 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     4441 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4501 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4561 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4621 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4681 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg
     4741 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     4801 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     4861 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4921 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4981 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5041 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5101 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5161 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5221 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5281 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5341 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     5401 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5461 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5521 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5581 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5641 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5701 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5761 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5821 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5881 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5941 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6001 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6061 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6121 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6181 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6241 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6301 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6361 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6421 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6481 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6541 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6601 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6661 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6721 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6781 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6841 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6901 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6961 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7021 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7081 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7141 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7201 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     7261 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     7321 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     7381 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     7441 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     7501 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     7561 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaag
//
