LOCUS       sequence-354125-        8780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for expressing U6 MS2-sgRNA cloning plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8780)
  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 8780)
  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_zhang2.0
FEATURES             Location/Qualifiers
     source          1..8780
                     /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"
     regulatory      1161..1170
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1167..1565
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    1589..2177
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1642..1662)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2060..2071)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2384..3017
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3146..3162)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3146..3162)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3159..3181)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3170..3186
                     /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(3194..3200,3201..3218,3219..3224))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3239..3260
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3377..3394)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3548..4136)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3628..3647)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4307..5098,5099..5167))
                     /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     4930..4949
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5168..5272)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    5320..5454
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5357..5376)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5411..5430
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5622..6255
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    6302..6427
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6924..7157
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7342..7386
                     /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    7649..7764
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        7867..8115
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7867..7887
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8038..8057
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8154..8172
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        8224..8242
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     enhancer        8346..8649
                     /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 gccaccatgg ccaagccttt gtctcaagaa gaatccaccc
     1201 tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg
     1261 ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta
     1321 ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca
     1381 acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac
     1441 ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg
     1501 atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg
     1561 gctaatgtac aagtaaagcg tctggaacaa tcaacctctg gattacaaaa tttgtgaaag
     1621 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     1681 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     1741 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     1801 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     1861 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     1921 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     1981 gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     2041 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     2101 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     2161 ttgggccgcc tccccgcctg gaattaattc tgcagtcgag acctagaaaa acatggagca
     2221 atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag
     2281 gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag
     2341 gcagctgtag atcttagcca ctttttaaaa gaaaagaggg gactggaagg gctaattcac
     2401 tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct
     2461 gattagcaga actacacacc agggccaggg gtcagatatc cactgacctt tggatggtgc
     2521 tacaagctag taccagttga gccagataag gtagaagagg ccaataaagg agagaacacc
     2581 agcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtgttagag
     2641 tggaggtttg acagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac
     2701 ttcaagaact gctgatatcg agcttgctac aagggacttt ccgctgggga ctttccaggg
     2761 aggcgtggcc tgggcgggac tggggagtgg cgagccctca gatcctgcat ataagcagct
     2821 gctttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     2881 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     2941 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     3001 gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc
     3061 aaagaaatga atatcagaga gtgagaggcc ttgacattgc tagcgtttta ccgtcgacct
     3121 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3181 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3241 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3301 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3361 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3421 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3481 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3541 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3601 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3661 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3721 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3781 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3841 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3901 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3961 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4021 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4081 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4141 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4201 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4261 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4321 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4381 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4441 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4501 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4561 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4621 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4681 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4741 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4801 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4861 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4921 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4981 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5041 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5101 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5161 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5221 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5281 cccgaaaagt gccacctgac gtcgacggat cgggagatca acttgtttat tgcagcttat
     5341 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5401 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcaactgg
     5461 ataactcaag ctaaccaaaa tcatcccaaa cttcccaccc cataccctat taccactgcc
     5521 aattacctgt ggtttcattt actctaaacc tgtgattcct ctgaattatt ttcattttaa
     5581 agaaattgta tttgttaaat atgtactaca aacttagtag ttggaagggc taattcactc
     5641 ccaaagaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga
     5701 ttagcagaac tacacaccag ggccaggggt cagatatcca ctgacctttg gatggtgcta
     5761 caagctagta ccagttgagc cagataaggt agaagaggcc aataaaggag agaacaccag
     5821 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tgttagagtg
     5881 gaggtttgac agccgcctag catttcatca cgtggcccga gagctgcatc cggagtactt
     5941 caagaactgc tgatatcgag cttgctacaa gggactttcc gctggggact ttccagggag
     6001 gcgtggcctg ggcgggactg gggagtggcg agccctcaga tcctgcatat aagcagctgc
     6061 tttttgcctg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     6121 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     6181 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     6241 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
     6301 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
     6361 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
     6421 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
     6481 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
     6541 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
     6601 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
     6661 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
     6721 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg gccgctgatc
     6781 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     6841 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     6901 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     6961 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     7021 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     7081 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     7141 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     7201 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     7261 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     7321 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     7381 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     7441 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     7501 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc
     7561 gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag atccattcga
     7621 ttagtgaacg gatctcgacg gtatcgcctt taaaagaaaa ggggggattg gggggtacag
     7681 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     7741 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccagttta
     7801 tcattagtga acggatctcg acggtatcga tcacgagact agcctcgagc ggccgccccc
     7861 ttcaccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     7921 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     7981 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     8041 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     8101 aaaggacgaa acaccggaga cgattaatgc gtctcggttt tagagctagg ccaacatgag
     8161 gatcacccat gtctgcaggg cctagcaagt taaaataagg ctagtccgtt atcaacttgg
     8221 ccaacatgag gatcacccat gtctgcaggg ccaagtggca ccgagtcggt gctttttttg
     8281 aattctcgac ctcgagacaa atggcagtat tcgtcattag ttcatagccc atatatggag
     8341 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     8401 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     8461 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     8521 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     8581 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     8641 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
     8701 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     8761 ggggggggcg cgcgccaggc
//
