LOCUS       sequence-338362-        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        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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        1713..1961
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1713..1733
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1884..1903
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1980..2055
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2103..2220
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2278..2684
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2719..2728
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2725..2727,2728..2730,2731..3444)
                     /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(2725..2727,2728..2730,2731..3441)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2770..2791)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3031..3050)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3378..3399
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3460..4048
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3513..3533)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3931..3942)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4120..4353
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4431..4552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4468..4487)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4522..4541
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4592..4727
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4747..4766)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4748..4766)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4776..4793)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4776..4792)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4785..4807)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4934..5389
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5021..5040)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5231..5252
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5415..5519
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5520..5588,5589..6380)
                     /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(5738..5757)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6551..7139
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7040..7059
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7293..7310
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7427..7448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7463..7468,7469..7486,7487..7493)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7501..7517
                     /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     7506..7528
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7525..7541
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7525..7541
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7560..7580
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7562..7580
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggaccca gagagggcct atttcccatg attccttcat
     1741 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
     1801 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1861 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     1921 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggagacgtg accgtctctg
     1981 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     2041 gcaccgagtc ggtgcttttt tgaattctag atcttgagac aaatggcagt attcatccac
     2101 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     2161 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     2221 cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgagct agctgcagta
     2281 acgccatttt gcaaggcatg gaaaaatacc aaaccaagaa tagagaagtt cagatcaagg
     2341 gcgggtacat gaaaatagct aacgttgggc caaacaggat atctgcggtg agcagtttcg
     2401 gccccggccc ggggccaaga acagatggtc accgcagttt cggccccggc ccgaggccaa
     2461 gaacagatgg tccccagata tggcccaacc ctcagcagtt tcttaagacc catcagatgt
     2521 ttccaggctc ccccaaggac ctgaaatgac cctgcgcctt atttgaatta accaatcagc
     2581 ctgcttctcg cttctgttcg cgcgcttctg cttcccgagc tctataaaag agctcacaac
     2641 ccctcactcg gcgcgccagt cctccgacag actgagtcgg ccggtcgaat caagcttatc
     2701 gataccgtcg acggatccgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     2761 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     2821 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2881 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     2941 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     3001 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     3061 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     3121 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     3181 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     3241 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3301 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     3361 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3421 ggcatggacg agctgtacaa gtaaacgcgt taagtcgaca atcaacctct ggattacaaa
     3481 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     3541 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     3601 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     3661 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     3721 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     3781 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     3841 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     3901 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     3961 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     4021 cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag
     4081 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     4141 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     4201 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     4261 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     4321 gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta
     4381 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     4441 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4501 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4561 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4621 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4681 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
     4741 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     4801 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     4861 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     4921 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4981 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     5041 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5101 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5161 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5221 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5281 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5341 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5401 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5461 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5521 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5581 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5641 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5701 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5761 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5821 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5881 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5941 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6001 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6061 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6121 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6181 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6241 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6301 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6361 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6421 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6481 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6541 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6601 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6661 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6721 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6781 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6841 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6901 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6961 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     7021 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     7081 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7141 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     7201 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     7261 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     7321 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     7381 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     7441 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     7501 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
     7561 caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaa
//
