LOCUS       sequence-362096-        8726 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 8726)
  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 8726)
  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 Blasto
FEATURES             Location/Qualifiers
     source          1..8726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          123..1139
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1063..1085
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1147..1166
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1167..1176
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1173..1571
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    1595..2183
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1648..1668)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2066..2077)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2390..3023
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3152..3168)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3152..3168)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3165..3187)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3176..3192
                     /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(3200..3206,3207..3224,3225..3230))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3245..3266
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3383..3400)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3554..4142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3634..3653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4313..5104,5105..5173))
                     /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     4936..4955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5174..5278)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    5326..5460
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5363..5382)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5417..5436
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5628..6261
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    6308..6433
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6930..7163
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7348..7392
                     /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    7655..7770
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        7873..8121
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7873..7893
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8044..8063
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8143..8218
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8292..8595
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
       61 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      121 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      181 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      241 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      301 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      361 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      421 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
      481 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
      541 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
      601 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
      661 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
      721 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
      781 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
      841 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
      901 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
      961 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1021 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1081 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1141 tcctgggcaa cgtgctggtt attgtggcca ccatggccaa gcctttgtct caagaagaat
     1201 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     1261 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     1321 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     1381 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     1441 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
     1501 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     1561 gggagggcta atgtacaagt aaagcgtctg gaacaatcaa cctctggatt acaaaatttg
     1621 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     1681 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     1741 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     1801 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     1861 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     1921 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     1981 gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg
     2041 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     2101 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     2161 ctccctttgg gccgcctccc cgcctggaat taattctgca gtcgagacct agaaaaacat
     2221 ggagcaatca caagtagcaa tacagcagct accaatgctg attgtgcctg gctagaagca
     2281 caagaggagg aggaggtggg ttttccagtc acacctcagg tacctttaag accaatgact
     2341 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa agaggggact ggaagggcta
     2401 attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac
     2461 ttccctgatt agcagaacta cacaccaggg ccaggggtca gatatccact gacctttgga
     2521 tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa taaaggagag
     2581 aacaccagct tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagtg
     2641 ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga gctgcatccg
     2701 gagtacttca agaactgctg atatcgagct tgctacaagg gactttccgc tggggacttt
     2761 ccagggaggc gtggcctggg cgggactggg gagtggcgag ccctcagatc ctgcatataa
     2821 gcagctgctt tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct
     2881 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     2941 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     3001 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata
     3061 acttgcaaag aaatgaatat cagagagtga gaggccttga cattgctagc gttttaccgt
     3121 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3181 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3241 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3301 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3361 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3421 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3481 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3541 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3601 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3661 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3721 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3781 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3841 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3901 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3961 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4021 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4081 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4141 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4201 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4261 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4321 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4381 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4441 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4501 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4561 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4621 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4681 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4741 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4801 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4861 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4921 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4981 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5041 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5101 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5161 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5221 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5281 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatcaactt gtttattgca
     5341 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5401 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     5461 aactggataa ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc
     5521 actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga attattttca
     5581 ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagttgg aagggctaat
     5641 tcactcccaa agaagacaag atatccttga tctgtggatc taccacacac aaggctactt
     5701 ccctgattag cagaactaca caccagggcc aggggtcaga tatccactga cctttggatg
     5761 gtgctacaag ctagtaccag ttgagccaga taaggtagaa gaggccaata aaggagagaa
     5821 caccagcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtgtt
     5881 agagtggagg tttgacagcc gcctagcatt tcatcacgtg gcccgagagc tgcatccgga
     5941 gtacttcaag aactgctgat atcgagcttg ctacaaggga ctttccgctg gggactttcc
     6001 agggaggcgt ggcctgggcg ggactgggga gtggcgagcc ctcagatcct gcatataagc
     6061 agctgctttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     6121 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     6181 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     6241 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
     6301 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
     6361 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
     6421 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
     6481 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     6541 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     6601 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     6661 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     6721 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg
     6781 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     6841 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     6901 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     6961 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     7021 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     7081 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     7141 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     7201 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     7261 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     7321 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     7381 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     7441 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     7501 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     7561 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     7621 attcgattag tgaacggatc tcgacggtat cgcctttaaa agaaaagggg ggattggggg
     7681 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     7741 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     7801 agtttatcat tagtgaacgg atctcgacgg tatcgatcac gagactagcc tcgagcggcc
     7861 gcccccttca ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag
     7921 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     7981 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     8041 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     8101 ttgtggaaag gacgaaacac cggagacgat taatgcgtct cggttttaga gctagaaata
     8161 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     8221 tttttgaatt ctcgacctcg agacaaatgg cagtattcgt cattagttca tagcccatat
     8281 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     8341 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     8401 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     8461 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     8521 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     8581 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     8641 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     8701 gcgggggggg ggggcgcgcg ccaggc
//
