LOCUS       sequence-354127-        8913 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 8913)
  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 8913)
  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_TtoC and hairpin extension
FEATURES             Location/Qualifiers
     source          1..8913
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        complement(599..847)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(657..676)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(827..847)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    950..1065
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     CDS             complement(1328..1372)
                     /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    1557..1790
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2287..2412
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             2459..3092
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     polyA_signal    3260..3394
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3284..3303)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3338..3357
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3442..3546
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3547..3615,3616..4407)
                     /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(3765..3784)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4578..5166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5067..5086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5320..5337
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5454..5475
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5490..5495,5496..5513,5514..5520)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5528..5544
                     /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     5533..5555
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5552..5568
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5552..5568
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     LTR             5697..6330
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    6537..7125
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6643..6654
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7052..7072
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7149..7748)
                     /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(7232..7252)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     7729..7748
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(7755..7774)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          7782..8798
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(7836..7858)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 cccccccgcc cccatcgctg cacaaaataa ttaaaaaata aataaataca aaattggggg
       61 tggggagggg ggggagatgg ggagagtgaa gcagaacgtg gggctcacct cgaccatggt
      121 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
      181 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
      241 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
      301 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
      361 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
      421 gaactccata tatgggctat gaactaatga cgaatactgc catttgtctc gaggtcgaga
      481 attcaaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt
      541 taaacttgct atgctgtttc cagcatagct cttaaaccga gacgcattaa tcgtctccgg
      601 tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg
      661 taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt
      721 attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc
      781 caattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata
      841 ggccctcggt gaagggggcg gccgctcgag gctagtctcg tgatcgatac cgtcgagatc
      901 cgttcactaa tgataaactg gatctctgct gtccctgtaa taaacccgaa aattttgaat
      961 ttttgtaatt tgtttttgta attctttagt ttgtatgtct gttgctatta tgtctactat
     1021 tctttcccct gcactgtacc ccccaatccc cccttttctt ttaaaggcga taccgtcgag
     1081 atccgttcac taatcgaatg gatctgtctc tgtctctctc tccaccttct tcttctattc
     1141 cttcgggcct gtcgggtccc ctcggggttg ggaggtgggt ctgaaacgat aatggtgaat
     1201 atccctgcct aactctattc actatagaaa gtacagcaaa aactattctt aaacctacca
     1261 agcctcctac tatcattatg aataatttta tataccacag ccaatttgtt atgttaaacc
     1321 aattccacaa acttgcccat ttatctaatt ccaataattc ttgttcattc ttttcttgct
     1381 ggttttgcga ttcttcaatt aaggagtgta ttaagcttgt gtaattgtta atttctctgt
     1441 cccactccat ccaggtcgtg tgattccaaa tctgttccag agatttatta ctccaactag
     1501 cattccaagg cacagcagtg gtgcaaatga gttttccaga gcaaccccaa atccccagga
     1561 gctgttgatc ctttaggtat ctttccacag ccaggattct tgcctggagc tgcttgatgc
     1621 cccagactgt gagttgcaac agatgctgtt gcgcctcaat agccctcagc aaattgttct
     1681 gctgctgcac tataccagac aataattgtc tggcctgtac cgtcagcgtc attgacgctg
     1741 cgcccatagt gcttcctgct gctcccaaga acccaaggaa caaagctcct attcccactg
     1801 ctcttttttc tctctgcacc actcttctct ttgccttggt gggtgctact cctaatggtt
     1861 caatttttac tactttatat ttatataatt cacttctcca attgtccctc atatctcctc
     1921 ctccaggtct gaagatcagc ggccggccgc ttgctgtgcg gtggtcttac ttttgttttg
     1981 ctcttcctct atcttgtcta aagcttcctt ggtgtctttt atctctatcc tttgatgcac
     2041 acaatagagg gttgctactg tattatataa tgatctaagt tcttctgatc ctgtctgaag
     2101 ggatggttgt agctgtccca gtatttgtct acagccttct gatgtttcta acaggccagg
     2161 attaactgcg aatcgttcta gctccctgct tgcccatact atatgtttta atttatattt
     2221 tttctttccc cctggcctta accgaatttt ttcccatcgc gatctaattc tcccccgctt
     2281 aatactgacg ctctcgcacc catctctctc cttctagcct ccgctagtca aaatttttgg
     2341 cgtactcacc agtcgccgcc cctcgcctct tgccgtgcgc gcttcagcaa gccgagtcct
     2401 gcgtcgagag agctcctctg gtttcccttt cgctttcaag tccctgttcg ggcgccactg
     2461 ctagagattt tccacactga ctaaaagggt ctgagggatc tctagttacc agagtcacac
     2521 aacagacggg cacacactac ttgaagcact caaggcaagc tttattgagg cttaagcagt
     2581 gggttcccta gttagccaga gagctcccag gctcagatct ggtctaacca gagagaccca
     2641 gtacaggcaa aaagcagctg cttatatgca ggatctgagg gctcgccact ccccagtccc
     2701 gcccaggcca cgcctccctg gaaagtcccc agcggaaagt cccttgtagc aagctcgata
     2761 tcagcagttc ttgaagtact ccggatgcag ctctcgggcc acgtgatgaa atgctaggcg
     2821 gctgtcaaac ctccactcta acacttctct ctccgggtca tccatcccat gcaggctcac
     2881 agggtgtaac aagctggtgt tctctccttt attggcctct tctaccttat ctggctcaac
     2941 tggtactagc ttgtagcacc atccaaaggt cagtggatat ctgacccctg gccctggtgt
     3001 gtagttctgc taatcaggga agtagccttg tgtgtggtag atccacagat caaggatatc
     3061 ttgtcttctt tgggagtgaa ttagcccttc caactactaa gtttgtagta catatttaac
     3121 aaatacaatt tctttaaaat gaaaataatt cagaggaatc acaggtttag agtaaatgaa
     3181 accacaggta attggcagtg gtaatagggt atggggtggg aagtttggga tgattttggt
     3241 tagcttgagt tatccagttg atccagacat gataagatac attgatgagt ttggacaaac
     3301 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     3361 atttgtaacc attataagct gcaataaaca agttgatctc ccgatccgtc gacgtcaggt
     3421 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3481 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3541 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3601 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3661 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3721 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3781 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3841 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3901 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3961 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4021 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4081 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4141 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4201 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4261 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4321 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4381 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4441 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4501 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4561 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4621 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4681 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4741 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4801 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4861 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4921 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4981 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5041 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5101 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5161 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5221 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5281 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5341 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5401 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     5461 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5521 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     5581 aagctctagc tagaggtcga cggtaaaacg ctagcaatgt caaggcctct cactctctga
     5641 tattcatttc tttgcaagtt ataaatactg aataataaga tgacatgaac tactactgct
     5701 agagattttc cacactgact aaaagggtct gagggatctc tagttaccag agtcacacaa
     5761 cagacgggca cacactactt gaagcactca aggcaagctt tattgaggct taagcagtgg
     5821 gttccctagt tagccagaga gctcccaggc tcagatctgg tctaaccaga gagacccagt
     5881 acaggcaaaa agcagctgct tatatgcagg atctgagggc tcgccactcc ccagtcccgc
     5941 ccaggccacg cctccctgga aagtccccag cggaaagtcc cttgtagcaa gctcgatatc
     6001 agcagttctt gaagtactcc ggatgcagct ctcgggccac gtgatgaaat gctaggcggc
     6061 tgtcaaacct ccactctaac acttctctct ccgggtcatc catcccatgc aggctcacag
     6121 ggtgtaacaa gctggtgttc tctcctttat tggcctcttc taccttatct ggctcaactg
     6181 gtactagctt gtagcaccat ccaaaggtca gtggatatct gacccctggc cctggtgtgt
     6241 agttctgcta atcagggaag tagccttgtg tgtggtagat ccacagatca aggatatctt
     6301 gtcttcgttg ggagtgaatt agcccttcca gtcccctctt ttcttttaaa aagtggctaa
     6361 gatctacagc tgccttgtaa gtcattggtc ttaaaggtac ctgaggtgtg actggaaaac
     6421 ccacctcctc ctcctcttgt gcttctagcc aggcacaatc agcattggta gctgctgtat
     6481 tgctacttgt gattgctcca tgtttttcta ggtctcgact gcagaattaa ttccaggcgg
     6541 ggaggcggcc caaagggaga tccgactcgt ctgagggcga aggcgaagac gcggaagagg
     6601 ccgcagagcc ggcagcaggc cgcgggaagg aaggtccgct ggattgaggg ccgaagggac
     6661 gtagcagaag gacgtcccgc gcagaatcca ggtggcaaca caggcgagca gccatggaaa
     6721 ggacgtcagc ttccccgaca acaccacgga attgtcagtg cccaacagcc gagcccctgt
     6781 ccagcagcgg gcaaggcagg cggcgatgag ttccgccgtg gcaataggga gggggaaagc
     6841 gaaagtcccg gaaaggagct gacaggtggt ggcaatgccc caaccagtgg gggttgcgtc
     6901 agcaaacaca gtgcacacca cgccacgttg cctgacaacg ggccacaact cctcataaag
     6961 agacagcaac caggatttat acaaggagga gaaaatgaaa gccatacggg aagcaatagc
     7021 atgatacaaa ggcattaaag cagcgtatcc acatagcgta aaaggagcaa catagttaag
     7081 aataccagtc aatctttcac aaattttgta atccagaggt tgattgttcc agacgcttta
     7141 cttgtacatt aggcaccggg cttgcgggtc atgcaccagg tgcgcggtcc ttcgggcacc
     7201 tcgacgtcgg cggtgacggt gaagccgagc cgctcgtaga aggggaggtt gcggggcgcg
     7261 gaggtctcca ggaaggcggg caccccggcg cgctcggccg cctccactcc ggggagcacg
     7321 acggcgctgc ccagaccctt gccctggtgg tcgggcgaga ctccgacggt ggccaggaac
     7381 cacgcgggct ccttgggccg gtgcggcgcc aggaggcctt ccatctgttg ctgcgcggcc
     7441 agccgggaac cgctcaactc ggccatgcgc gggccgatct cggcgaacac cgcccccgct
     7501 tcgacgctct ccggcgtggt ccagaccgcc acggcggcgc cgtcgtccgc gacccacacc
     7561 ttgccgatgt cgagcccgac gcgcgtgagg aagagttctt gcagctcggt gacccgctcg
     7621 atgtggcggt ccggatcgac ggtgtggcgc gtggcggggt agtcggcgaa cgcggcggcg
     7681 agggtgcgta cggccctggg gacgtcgtcg cgggtggcga ggcgcaccgt gggcttgtac
     7741 tcggtcatgg tggccacaat aaccagcacg ttgcccagga gctgtaggaa aaagaagaag
     7801 gcatgaacat ggttagcaga ggctctagag ccgccggtca cacgccagaa gccgaacccc
     7861 gccctgcccc gtcccccccg aaggcagccg tccccccgcg gacagccccg aggctggaga
     7921 gggagaaggg gacggcggcg cggcgacgca cgaaggccct ccccgcccat ttccttcctg
     7981 ccggcgccgc accgcttcgc cccgcgcccg ctagaggggg tgcggcggcg cctcccagat
     8041 ttcggctccg cacagatttg ggacaaagga agtccctgcg ccctctcgca cgattaccat
     8101 aaaaggcaat ggctgcggct cgccgcgcct cgacagccgc cggcgctccg ggggccgccg
     8161 cgcccctccc ccgagccctc cccggcccga ggcggccccg ccccgcccgg cacccccacc
     8221 tgccgccacc ccccgcccgg cacggcgagc cccgcgccac gccccgtacg gagccccgca
     8281 cccgaagccg ggccgtgctc agcaactcgg ggaggggggt gcaggggggg ttgcagcccg
     8341 accgacgcgc ccacaccccc tgctcacccc cccacgcaca caccccgcac gcagcctttg
     8401 ttcccctcgc agcccccccc gcaccgcggg gcaccgcccc cggccgcgct cccctcgcgc
     8461 acactgcgga gcgcacaaag ccccgcgccg cgcccgcagc gctcacagcc gccgggcagc
     8521 gcggagccgc acgcggcgct ccccacgcac acacacacgc acgcaccccc cgagccgctc
     8581 cccccgcaca aagggccctc ccggagcccc tcaaggcttt cacgcagcca cagaaaagaa
     8641 acaagccgtc attaaaccaa gcgctaatta cagcccggag gagaagggcc gtcccgcccg
     8701 ctcacctgtg ggagtaacgc ggtcagtcag agccggggcg ggcggcgcga ggcggcggcg
     8761 gagcggggca cggggcgaag gcagcgcgca gcgactcccg cccgccgcgc gcttcgcttt
     8821 ttatagggcc gccgccgccg ccgcctcgcc ataaaaggaa actttcggag cgcgccgctc
     8881 tgattggctg ccgccgcacc tctccgcctc gcc
//
