LOCUS       sequence-102366-        8122 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An empty optimized gRNA expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8122)
  AUTHORS   Ochiai H, Sugawara T, Yamamoto T
  TITLE     Simultaneous live imaging of the transcription and nuclear position 
            of specific genes.
  JOURNAL   Nucleic Acids Res. 2015 Jun 19. pii: gkv624.
  PUBMED    26092696
REFERENCE   2  (bases 1 to 8122)
  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: "Nucleic 
            Acids Res. 2015 Jun 19. pii: gkv624."
COMMENT     Sequence Label: pKLV-U6gRNA-EF(BbsI)-PGKpuro2ABFP
FEATURES             Location/Qualifiers
     source          1..8122
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        715..1018
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1020..1218
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1173..1193
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1236..1416
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1463..1588
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2087..2320
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2505..2549
                     /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    2847..2964
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3015..3263
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3015..3035
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3186..3205
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3414..3913
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3449..3472)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3846..3865
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3953..4549
                     /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(3953..3972)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4449..4469
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4568..4621
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4622..5323
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(5678..5695)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5684..5908
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5934..5954)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5934..5952)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5973..5989)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5973..5989)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5986..6008)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5997..6013
                     /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(6021..6027,6028..6045,6046..6051))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6066..6087
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6204..6221)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6375..6963)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6455..6474)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7134..7925,7926..7994))
                     /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     7757..7776
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7995..8099)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgactagtta
      661 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      721 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      781 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      841 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      901 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      961 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1021 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     1081 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     1141 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     1201 ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct
     1261 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     1321 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     1381 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     1441 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     1501 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     1561 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     1621 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     1681 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     1741 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     1801 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     1861 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     1921 gcaagcggcc ggccgcgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     1981 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     2041 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     2101 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     2161 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     2221 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     2281 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     2341 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     2401 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     2461 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     2521 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     2581 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     2641 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     2701 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     2761 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     2821 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     2881 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     2941 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     3001 ttagtaccgg gcccgagggc ctatttccca tgattccttc atatttgcat atacgataca
     3061 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     3121 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     3181 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     3241 tcttgtggaa aggacgaaac accgggtctt cgagaagacc tgtttaagag ctatgctgga
     3301 aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     3361 tcggtgcttt tttgttttag agctagaaat agcaagttgg atccaattct accgggtagg
     3421 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     3481 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     3541 ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     3601 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     3661 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     3721 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     3781 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     3841 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     3901 tccgggcctt tcgacctgca tccatctaga tctcgagcag ctgaagctta ccatgaccga
     3961 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     4021 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     4081 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     4141 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     4201 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     4261 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     4321 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     4381 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     4441 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     4501 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gcggcgggtc
     4561 cggaggagag ggcagaggaa gtctcctaac atgcggtgac gtggaggaga atcctggccc
     4621 aatgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     4681 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     4741 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     4801 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     4861 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aggacggggg
     4921 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     4981 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     5041 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
     5101 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc
     5161 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     5221 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
     5281 atactgcgac ctccctagca aactggggca caagcttaat tgagcggccg ctaggtacct
     5341 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     5401 ggactggaag ggctaattca ctcccaaaga agtcaagatc tgctttttgc ctgtactggg
     5461 tctctctggt tagaccagag tctctctggt tagaccagat ctgagcctgg gagctctctg
     5521 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     5581 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     5641 tgtggaaaat ctctagcagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     5701 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     5761 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     5821 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaagt caatagcagg
     5881 catgctgggg atgcggtggg ctctatgggc ggccgttaat taagcttttg ttccctttag
     5941 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     6001 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     6061 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6121 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6181 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     6241 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     6301 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     6361 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     6421 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     6481 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     6541 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6601 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6661 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6721 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6781 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     6841 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6901 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6961 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     7021 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     7081 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     7141 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     7201 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     7261 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     7321 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     7381 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     7441 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     7501 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     7561 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7621 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7681 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7741 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7801 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7861 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7921 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7981 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     8041 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     8101 acatttcccc gaaaagtgcc ac
//
