LOCUS       sequence-354122-        8908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for expressing U6 sgRNA cloning plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8908)
  AUTHORS   Nghi Dang , Alissa Lance-Byrne, Angela Tung, Xiaoge Guo, Ryan J 
            Cecchi, Joanna Buchthal, Alejandro Chavez, Nan Cher Yeo
  TITLE     Generation and application of a versatile CRISPR toolkit for 
            mammalian cell engineering
  JOURNAL   Synthetic Biology, Volume 6, Issue 1, 2021
REFERENCE   2  (bases 1 to 8908)
  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"; journalName: "Synthetic 
            Biology, Volume 6, Issue 1, 2021"
COMMENT     Sequence Label: pSB700 lenti gRNA Puro
FEATURES             Location/Qualifiers
     source          1..8908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          117..1133
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1057..1079
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1141..1160
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1167..1766
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1167..1186)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1663..1683
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1790..2378
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1843..1863)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2261..2272)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2585..3218
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3347..3363)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3347..3363)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3360..3382)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3371..3387
                     /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)."
     promoter        complement(join(3395..3401,3402..3419,3420..3425))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3440..3461
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3578..3595)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3749..4337)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3829..3848)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4508..5299,5300..5368))
                     /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     5131..5150
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5369..5473)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    5521..5655
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5558..5577)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5612..5631
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5823..6456
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    6503..6628
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7125..7358
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7543..7587
                     /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"
     misc_feature    7850..7965
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        8068..8316
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8068..8088
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8239..8258
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8338..8413
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8487..8790
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
       61 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
      121 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
      181 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
      241 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc
      301 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt
      361 gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg
      421 ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg
      481 gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
      541 tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc
      601 ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg
      661 cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc
      721 cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct
      781 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
      841 gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct
      901 ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc
      961 ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg
     1021 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1081 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1141 gcaacgtgct ggttattgtg gccaccatga ccgagtacaa gcccacggtg cgcctcgcca
     1201 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     1261 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     1321 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     1381 ccgtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     1441 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     1501 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc
     1561 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     1621 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     1681 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     1741 gcatgacccg caagcccggt gcctaatgta caagtaaagc gtctggaaca atcaacctct
     1801 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     1861 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     1921 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     1981 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     2041 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     2101 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     2161 caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc
     2221 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     2281 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     2341 tcagacgagt cggatctccc tttgggccgc ctccccgcct ggaattaatt ctgcagtcga
     2401 gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt
     2461 gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct
     2521 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagagg
     2581 ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac
     2641 cacacacaag gctacttccc tgattagcag aactacacac cagggccagg ggtcagatat
     2701 ccactgacct ttggatggtg ctacaagcta gtaccagttg agccagataa ggtagaagag
     2761 gccaataaag gagagaacac cagcttgtta caccctgtga gcctgcatgg gatggatgac
     2821 ccggagagag aagtgttaga gtggaggttt gacagccgcc tagcatttca tcacgtggcc
     2881 cgagagctgc atccggagta cttcaagaac tgctgatatc gagcttgcta caagggactt
     2941 tccgctgggg actttccagg gaggcgtggc ctgggcggga ctggggagtg gcgagccctc
     3001 agatcctgca tataagcagc tgctttttgc ctgtactggg tctctctggt tagaccagat
     3061 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     3121 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     3181 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat
     3241 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagaggc cttgacattg
     3301 ctagcgtttt accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     3361 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3421 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3481 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3541 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3601 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3661 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3721 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3781 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3841 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3901 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3961 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4021 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4081 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4141 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4201 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4261 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4321 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4381 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4441 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4501 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4561 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4621 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4681 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4741 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4801 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4861 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4921 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4981 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5041 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5101 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5161 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5221 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5281 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5341 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5401 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5461 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     5521 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     5581 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5641 tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc
     5701 ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac ctgtgattcc
     5761 tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac aaacttagta
     5821 gttggaaggg ctaattcact cccaaagaag acaagatatc cttgatctgt ggatctacca
     5881 cacacaaggc tacttccctg attagcagaa ctacacacca gggccagggg tcagatatcc
     5941 actgaccttt ggatggtgct acaagctagt accagttgag ccagataagg tagaagaggc
     6001 caataaagga gagaacacca gcttgttaca ccctgtgagc ctgcatggga tggatgaccc
     6061 ggagagagaa gtgttagagt ggaggtttga cagccgccta gcatttcatc acgtggcccg
     6121 agagctgcat ccggagtact tcaagaactg ctgatatcga gcttgctaca agggactttc
     6181 cgctggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag
     6241 atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct
     6301 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     6361 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     6421 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     6481 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     6541 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     6601 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     6661 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     6721 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     6781 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     6841 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     6901 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     6961 gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     7021 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     7081 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     7141 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     7201 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     7261 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     7321 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     7381 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     7441 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     7501 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     7561 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     7621 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     7681 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     7741 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga
     7801 gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcct ttaaaagaaa
     7861 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     7921 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     7981 ggacagcaga gatccagttt atcattagtg aacggatctc gacggtatcg atcacgagac
     8041 tagcctcgag cggccgcccc cttcaccgag ggcctatttc ccatgattcc ttcatatttg
     8101 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     8161 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     8221 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     8281 ttggctttat atatcttgtg gaaaggacga aacaccggag acgattaatg cgtctcggtt
     8341 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     8401 accgagtcgg tgcttttttt gaattctcga cctcgagaca aatggcagta ttcgtcatta
     8461 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     8521 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     8581 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     8641 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     8701 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     8761 atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact
     8821 ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
     8881 tgtgcagcga tgggggcggg gggggggg
//
