LOCUS       sequence-108236-        8801 bp    DNA     circular SYN 14-APR-2025
DEFINITION  An empty sgRNA expression vector for expression of sgRNA for Sp 
            Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8801)
  AUTHORS   Pham H, Kearns NA, Maehr R
  TITLE     Transcriptional Regulation with CRISPR/Cas9 Effectors in Mammalian 
            Cells.
  JOURNAL   Methods Mol Biol. 2016;1358:43-57. doi: 10.1007/978-1-4939-3067-8_3.
  PUBMED    26463376
REFERENCE   2  (bases 1 to 8801)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods Mol
            Biol."; date: "2016"; volume: "1358:43-57. doi"; pages: " 
            10.1007/978-1-4939-3067-8_3"
COMMENT     Sequence Label: pLenti SpBsmBI sgRNA Hygro
FEATURES             Location/Qualifiers
     source          1..8801
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..235
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             236..416
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    463..588
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1081..1314
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1499..1543
                     /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_RNA        complement(1822..1897)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1917..2165)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1975..1994)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2145..2165)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2196..2312
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2516..3698
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2747..3689
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3646..3666
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    4653..5241
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4706..4726)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5124..5135)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5328..5561
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5633..5754
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5670..5689)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5724..5743
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5794..5929
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5949..5968)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5950..5968)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5978..5995)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5978..5994)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5987..6009)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6136..6591
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6223..6242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6433..6454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6617..6721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6722..6790,6791..7582)
                     /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(6940..6959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7753..8341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8242..8261
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8495..8512
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8629..8650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8665..8670,8671..8688,8689..8695)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8703..8719
                     /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     8708..8730
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8727..8743
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8727..8743
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8762..8782
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8764..8782
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
       61 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
      121 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
      181 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
      241 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      301 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      361 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      421 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      481 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
      541 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
      601 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
      661 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
      721 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
      781 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
      841 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
      901 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
      961 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1021 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1081 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1141 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1201 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1261 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     1321 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     1381 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     1441 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     1501 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     1561 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     1621 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     1681 atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga
     1741 agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta
     1801 tcggttaaca ctagaaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac
     1861 tagccttatt ttaacttgct atttctagct ctaaaacgag acgcattaat cgtctcggtg
     1921 tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca agttacggta
     1981 agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc aagaaattat
     2041 tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa attaattcta
     2101 attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat gggaaatagg
     2161 ccctcttcct gcccgacctt ggtaccggat ccaactttta aaagaaaagg ggggattggg
     2221 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     2281 ttacaaaaac aaattacaaa attcaaaatt ttatcgatgc ctccccgtca ccaccccccc
     2341 caacccgccc cgaccggagc tgagagtaat tcatacaaaa ggactcgccc ctgccttggg
     2401 gaatcccagg gaccgtcgtt aaactcccac taacgtagaa cccagagatc gctgcgttcc
     2461 cgccccctca cccgcccgct ctcgtcatca ctgaggtgga gaagagcatg cgtgaggctc
     2521 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     2581 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     2641 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     2701 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
     2761 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
     2821 cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     2881 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     2941 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     3001 tcgataagtc tctagccatt taaaattttt gatgatatcc tgcgacgctt tttttctggc
     3061 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     3121 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     3181 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     3241 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     3301 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     3361 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     3421 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     3481 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     3541 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     3601 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     3661 tggttcaaag tttttttctt ccatttcagg tgtcgtgaaa actacccctc tagtgccacc
     3721 atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     3781 agcgtcagcg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     3841 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     3901 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     3961 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     4021 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     4081 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     4141 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     4201 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     4261 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     4321 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     4381 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     4441 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     4501 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     4561 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     4621 gccgggactg tcgggcgtac acaaatctcg acaatcaacc tctggattac aaaatttgtg
     4681 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     4741 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     4801 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     4861 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     4921 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     4981 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     5041 cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg
     5101 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     5161 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     5221 ccctttgggc cgcctccccg cctggaattc gagctcggta cctttaagac caatgactta
     5281 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     5341 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
     5401 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     5461 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     5521 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct
     5581 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     5641 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5701 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5761 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     5821 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     5881 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     5941 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     6001 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     6061 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     6121 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     6181 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     6241 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     6301 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     6361 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     6421 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     6481 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     6541 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     6601 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6661 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     6721 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     6781 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     6841 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     6901 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6961 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     7021 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     7081 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     7141 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     7201 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     7261 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     7321 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     7381 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     7441 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     7501 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     7561 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     7621 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     7681 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7741 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     7801 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     7861 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     7921 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7981 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     8041 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     8101 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     8161 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     8221 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     8281 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     8341 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     8401 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     8461 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     8521 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     8581 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     8641 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     8701 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     8761 cgcaattaac cctcactaaa gggaacaaaa gctggagctg c
//
