LOCUS       sequence-329211-        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        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"
     CDS             2724..3425
                     /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    3441..4029
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3494..3514)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3912..3923)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4101..4334
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4412..4533
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4449..4468)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4503..4522
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4573..4708
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4728..4747)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4729..4747)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4757..4774)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4757..4773)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4766..4788)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4915..5370
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5002..5021)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5212..5233
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5396..5500
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5501..5569,5570..6361)
                     /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(5719..5738)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6532..7120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7021..7040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7274..7291
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7408..7429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7444..7449,7450..7467,7468..7474)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7482..7498
                     /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     7487..7509
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7506..7522
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7506..7522
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7541..7561
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7543..7561
                     /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 tccatgagcg agctgatcac cgagaacatg cacatgaagc
     2761 tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca
     2821 agccctacga gggcacccag acccagagaa tcaaggtggt cgagggcggc cctctcccct
     2881 tcgccttcga catcctggct accagcttca tgtacggcag tcacaccttc atcaaccaca
     2941 cccagggcat ccccgacttc tggaagcagt ccttccctga gggcttcaca tgggagagag
     3001 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     3061 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga
     3121 tgcagaagaa aacactcggc tgggaggccc acaccgagat gctgtacccc gctgacggcg
     3181 gcctggaagg cagaaccgcg ctggccctga agctcgtggg cgggggccac ctgatctgca
     3241 acttcaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct
     3301 actatgtgga ctacagactg gaaagaatca aggaggccga caaagagacc tacgtcgagc
     3361 agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg cacaagctta
     3421 attaaacgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg
     3481 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     3541 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     3601 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     3661 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     3721 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     3781 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     3841 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     3901 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     3961 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     4021 cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     4081 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
     4141 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     4201 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     4261 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     4321 gaaaatctct agcagtacgt atagtagttc atgtcatctt attattcagt atttataact
     4381 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
     4441 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4501 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
     4561 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4621 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4681 aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt
     4741 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     4801 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     4861 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc
     4921 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4981 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5041 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5101 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     5161 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5221 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     5281 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5341 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     5401 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5461 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5521 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5581 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5641 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5701 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5761 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5821 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5881 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5941 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6001 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6061 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6121 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6181 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6241 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6301 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6361 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6421 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6481 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6541 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6601 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6661 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6721 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6781 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6841 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6901 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6961 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7021 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7081 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7141 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7201 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     7261 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     7321 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     7381 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     7441 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     7501 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
     7561 ggaacaaaag ctggagctgc aag
//
