LOCUS       sequence-338357-        7595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses S. pyogenes CRISPR chimeric RNA element with customizable 
            sgRNA from U6 promoter and tRFP 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 7595)
  TITLE     Ebert Lab plasmids
REFERENCE   2  (bases 1 to 7595)
  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.tRFP
FEATURES             Location/Qualifiers
     source          1..7595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     134..154
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        182..408
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             409..589
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    636..761
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1254..1487
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1672..1716
                     /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        1894..2142
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1894..1914
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2065..2084
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2161..2236
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2284..2401
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2459..2865
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             2900..3613
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    3629..4217
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3682..3702)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4100..4111)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4289..4522
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4600..4721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4637..4656)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4691..4710
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4761..4896
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4916..4935)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4917..4935)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4945..4962)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4945..4961)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4954..4976)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5103..5558
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5190..5209)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5400..5421
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5584..5688
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5689..5757,5758..6549)
                     /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(5907..5926)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6720..7308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7209..7228
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7462..7479
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
      181 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      241 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      301 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      361 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      421 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      481 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      541 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      601 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      661 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      721 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      781 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      841 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
      901 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
      961 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1021 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1081 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1141 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1201 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1261 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1321 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1381 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1441 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1501 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1561 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1621 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1681 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1741 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1801 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1861 cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat gattccttca
     1921 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
     1981 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     2041 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     2101 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggagacgt gaccgtctct
     2161 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     2221 ggcaccgagt cggtgctttt ttgaattcta gatcttgaga caaatggcag tattcatcca
     2281 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     2341 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     2401 tcgggtttat tacagggaca gcagagatcc actttggcgc cggctcgagc tagctgcagt
     2461 aacgccattt tgcaaggcat ggaaaaatac caaaccaaga atagagaagt tcagatcaag
     2521 ggcgggtaca tgaaaatagc taacgttggg ccaaacagga tatctgcggt gagcagtttc
     2581 ggccccggcc cggggccaag aacagatggt caccgcagtt tcggccccgg cccgaggcca
     2641 agaacagatg gtccccagat atggcccaac cctcagcagt ttcttaagac ccatcagatg
     2701 tttccaggct cccccaagga cctgaaatga ccctgcgcct tatttgaatt aaccaatcag
     2761 cctgcttctc gcttctgttc gcgcgcttct gcttcccgag ctctataaaa gagctcacaa
     2821 cccctcactc ggcgcgccag tcctccgaca gactgagtcg gccggtcgaa tcaagcttat
     2881 cgataccgtc gacggatcca tggtgtctaa gggcgaagag ctgattaagg agaacatgca
     2941 catgaagctg tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg
     3001 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
     3061 tctccccttc gccttcgaca tcctggctac cagcttcatg tacggcagca gaaccttcat
     3121 caaccacacc cagggcatcc ccgacttctt taagcagtcc ttccctgagg gcttcacatg
     3181 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     3241 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg
     3301 ccctgtgatg cagaagaaaa cactcggctg ggaggccaac accgagatgc tgtaccccgc
     3361 tgacggcggc ctggaaggca gaagcgacat ggccctgaag ctcgtgggcg ggggccacct
     3421 gatctgcaac ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc
     3481 cggcgtctac tatgtggacc acagactgga aagaatcaag gaggccgaca aagagaccta
     3541 cgtcgagcag cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca
     3601 caaacttaat tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag
     3661 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3721 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3781 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3841 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3901 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3961 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     4021 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     4081 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     4141 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     4201 ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt
     4261 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     4321 gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     4381 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     4441 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     4501 tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat
     4561 ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat
     4621 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4681 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta
     4741 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4801 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     4861 tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta
     4921 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     4981 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     5041 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     5101 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     5161 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     5221 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5281 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5341 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5401 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     5461 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     5521 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     5581 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5641 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5701 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5761 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5821 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5881 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5941 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     6001 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     6061 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     6121 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     6181 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     6241 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     6301 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     6361 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     6421 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6481 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6541 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6601 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6661 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6721 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6781 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6841 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6901 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6961 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     7021 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     7081 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     7141 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7201 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7261 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7321 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     7381 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     7441 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     7501 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     7561 cccgactgga aagcgggcag tgagcgcaac gcaat
//
