LOCUS       sequence-354516-        9043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for expressing U6 FE-sgRNA cloning plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9043)
  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 9043)
  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 EBFP2_TtoC and hairpin exten...
FEATURES             Location/Qualifiers
     source          1..9043
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          122..1138
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1062..1084
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1146..1165
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1166..1175
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1172..1174,1175..1177,1178..1891)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1217..1238)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    1915..2503
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1968..1988)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2386..2397)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2710..3343
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3472..3488)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3472..3488)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3485..3507)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3496..3512
                     /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(3520..3526,3527..3544,3545..3550))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3565..3586
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3703..3720)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3874..4462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3954..3973)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4633..5424,5425..5493))
                     /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     5256..5275
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5494..5598)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    5646..5780
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5683..5702)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5737..5756
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5948..6581
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    6628..6753
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7250..7483
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7668..7712
                     /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    7975..8090
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        8193..8441
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8193..8213
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8364..8383
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        8622..8925
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggcgggscg aggcggrgmg gkgcggcggc agccaatcag agcggcgcgc tccgaaagtt
       61 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      121 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      181 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      241 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      301 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      361 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      421 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
      481 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
      541 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
      601 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
      661 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
      721 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
      781 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
      841 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
      901 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
      961 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1021 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1081 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1141 cctgggcaac gtgctggtta ttgtggccac catggtgagc aagggcgagg agctgttcac
     1201 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1261 gaggggcgag ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac
     1321 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgagcc acggcgtgca
     1381 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1441 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcacct acaagacccg
     1501 cgccgaggtg aagttcgagg gcgacaccct agtgaaccgc atcgagctga agggcgtcga
     1561 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttca acagccacaa
     1621 catctatatc atggccgtca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1681 caacgtggag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1741 cgacggcccc gtgctgctgc ccgacagcca ctacctgagc acccagtccg tgctgagcaa
     1801 agaccccaac gagaagcgcg atcacatggt cctgctggag ttccgcaccg ccgccgggat
     1861 cactctcggc atggacgagc tgtacaagta atgtacaagt aaagcgtctg gaacaatcaa
     1921 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     1981 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     2041 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     2101 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     2161 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     2221 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     2281 actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct gctcgcctgt
     2341 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     2401 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     2461 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggaat taattctgca
     2521 gtcgagacct agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg
     2581 attgtgcctg gctagaagca caagaggagg aggaggtggg ttttccagtc acacctcagg
     2641 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     2701 agaggggact ggaagggcta attcactccc aacgaagaca agatatcctt gatctgtgga
     2761 tctaccacac acaaggctac ttccctgatt agcagaacta cacaccaggg ccaggggtca
     2821 gatatccact gacctttgga tggtgctaca agctagtacc agttgagcca gataaggtag
     2881 aagaggccaa taaaggagag aacaccagct tgttacaccc tgtgagcctg catgggatgg
     2941 atgacccgga gagagaagtg ttagagtgga ggtttgacag ccgcctagca tttcatcacg
     3001 tggcccgaga gctgcatccg gagtacttca agaactgctg atatcgagct tgctacaagg
     3061 gactttccgc tggggacttt ccagggaggc gtggcctggg cgggactggg gagtggcgag
     3121 ccctcagatc ctgcatataa gcagctgctt tttgcctgta ctgggtctct ctggttagac
     3181 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     3241 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     3301 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat
     3361 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggccttga
     3421 cattgctagc gttttaccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     3481 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3541 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3601 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3661 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3721 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3781 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3841 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3901 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3961 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4021 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4081 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4141 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4201 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4261 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4321 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4381 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4441 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4501 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4561 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4621 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4681 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4741 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4801 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4861 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4921 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4981 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5041 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5101 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5161 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5221 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5281 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5341 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5401 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5461 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5521 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5581 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     5641 agatcaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     5701 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5761 tatcttatca tgtctggatc aactggataa ctcaagctaa ccaaaatcat cccaaacttc
     5821 ccaccccata ccctattacc actgccaatt acctgtggtt tcatttactc taaacctgtg
     5881 attcctctga attattttca ttttaaagaa attgtatttg ttaaatatgt actacaaact
     5941 tagtagttgg aagggctaat tcactcccaa agaagacaag atatccttga tctgtggatc
     6001 taccacacac aaggctactt ccctgattag cagaactaca caccagggcc aggggtcaga
     6061 tatccactga cctttggatg gtgctacaag ctagtaccag ttgagccaga taaggtagaa
     6121 gaggccaata aaggagagaa caccagcttg ttacaccctg tgagcctgca tgggatggat
     6181 gacccggaga gagaagtgtt agagtggagg tttgacagcc gcctagcatt tcatcacgtg
     6241 gcccgagagc tgcatccgga gtacttcaag aactgctgat atcgagcttg ctacaaggga
     6301 ctttccgctg gggactttcc agggaggcgt ggcctgggcg ggactgggga gtggcgagcc
     6361 ctcagatcct gcatataagc agctgctttt tgcctgtact gggtctctct ggttagacca
     6421 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     6481 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     6541 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac
     6601 ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg
     6661 cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga
     6721 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg
     6781 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata
     6841 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca
     6901 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa
     6961 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata
     7021 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc
     7081 gcacagcaag cggccggccg ctgatcttca gacctggagg aggagatatg agggacaatt
     7141 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
     7201 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
     7261 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg
     7321 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
     7381 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
     7441 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
     7501 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
     7561 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca
     7621 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
     7681 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
     7741 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt
     7801 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga
     7861 cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag
     7921 agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat cgcctttaaa
     7981 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     8041 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     8101 tacagggaca gcagagatcc agtttatcat tagtgaacgg atctcgacgg tatcgatcac
     8161 gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat
     8221 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     8281 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     8341 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     8401 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggagacgat taatgcgtct
     8461 cggtttaaga gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca
     8521 acttgaaaaa gtggcaccga gtcggtgctt tttttgaatt ctcgacctcg agacaaatgg
     8581 cagtattcgt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     8641 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     8701 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     8761 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     8821 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     8881 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc
     8941 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt
     9001 attttttaat tattttgtgc agcgatgggg gcgggggggg ggg
//
