LOCUS       sequence-338364-        7583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses S. pyogenes CRISPR chimeric RNA element with customizable 
            sgRNA from U6 promoter and RFP657 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 7583)
  TITLE     Ebert Lab plasmids
REFERENCE   2  (bases 1 to 7583)
  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.RFP657
FEATURES             Location/Qualifiers
     source          1..7583
                     /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"
     CDS             2719..3420
                     /codon_start=1
                     /product="far-red monomeric fluorescent protein with an 
                     emission peak at 657 nm (Morozova et al., 2010)"
                     /label=TagRFP657
                     /note="derived from mKate"
                     /translation="MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQRIKV
                     VEGGPLPFAFDILATSFMYGSHTFINHTQGIPDFWKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEAHTEMLYPADGGLEGRTALALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDYRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    3436..4024
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3489..3509)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3907..3918)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4096..4329
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4407..4528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4444..4463)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4498..4517
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4568..4703
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4723..4742)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4724..4742)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4752..4769)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4752..4768)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4761..4783)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4910..5365
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4997..5016)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5207..5228
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5391..5495
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5496..5564,5565..6356)
                     /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(5714..5733)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6527..7115
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7016..7035
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7269..7286
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7403..7424
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7439..7444,7445..7462,7463..7469)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7477..7493
                     /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     7482..7504
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7501..7517
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7501..7517
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7536..7556
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7538..7556
                     /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 acggatccat gagcgagctg atcaccgaga acatgcacat gaagctgtac
     2761 atggagggca ccgtgaacaa ccaccacttc aagtgcacat ccgagggcga aggcaagccc
     2821 tacgagggca cccagaccca gagaatcaag gtggtcgagg gcggccctct ccccttcgcc
     2881 ttcgacatcc tggctaccag cttcatgtac ggcagtcaca ccttcatcaa ccacacccag
     2941 ggcatccccg acttctggaa gcagtccttc cctgagggct tcacatggga gagagtcacc
     3001 acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc
     3061 ctcatctaca acgtcaagat cagaggggtg aacttcccat ccaacggccc tgtgatgcag
     3121 aagaaaacac tcggctggga ggcccacacc gagatgctgt accccgctga cggcggcctg
     3181 gaaggcagaa ccgcgctggc cctgaagctc gtgggcgggg gccacctgat ctgcaacttc
     3241 aagaccacat acagatccaa gaaacccgct aagaacctca agatgcccgg cgtctactat
     3301 gtggactaca gactggaaag aatcaaggag gccgacaaag agacctacgt cgagcagcac
     3361 gaggtggctg tggccagata ctgcgacctc cctagcaaac tggggcacaa gcttaattaa
     3421 acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3481 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3541 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3601 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3661 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3721 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3781 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     3841 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3901 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     3961 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     4021 ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     4081 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     4141 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     4201 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     4261 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     4321 tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa
     4381 agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat
     4441 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4501 gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac
     4561 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4621 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4681 gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat
     4741 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     4801 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     4861 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     4921 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4981 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5041 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5101 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5161 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5221 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5281 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5341 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5401 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5461 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5521 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5581 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5641 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5701 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5761 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5821 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5881 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5941 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6001 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6061 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6121 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6181 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6241 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6301 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6361 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6421 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6481 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6541 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6601 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6661 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6721 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6781 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6841 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6901 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6961 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7021 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7081 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7141 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     7201 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     7261 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     7321 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     7381 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     7441 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     7501 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
     7561 aaaagctgga gctgcaagct taa
//
