LOCUS       sequence-258385-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses control gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8877)
  AUTHORS   Kleaveland B, Shi CY, Stefano J, Bartel DP
  TITLE     A Network of Noncoding Regulatory RNAs Acts in the Mammalian Brain.
  JOURNAL   Cell. 2018 Jul 12;174(2):350-362.e17. doi: 
            10.1016/j.cell.2018.05.022. Epub 2018 Jun 7.
  PUBMED    29887379
REFERENCE   2  (bases 1 to 8877)
  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"; doi: 
            "10.1016/j.cell.2018.05.022"; journalName: "Cell"; date: 
            "2018-07-12- 12"; volume: "174"; issue: "2"; pages: "350-362.e17"
COMMENT     Sequence Label: pU6-sgRNA EF1Alpha-puro-T2A-BFP.Control
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        81..279
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     234..254
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             297..477
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    524..649
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1146..1379
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1564..1608
                     /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    1906..2023
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2276..2309)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2313..2333
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        2578..2789
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          2790..3728
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3685..3705
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3749..4345
                     /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(3749..3768)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4245..4265
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4355..4408
                     /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             4421..5119
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5139..5172)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5228..5816
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5281..5301)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5699..5710)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5819..5835)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5820..5836)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6345..6525
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6587..7175)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6667..6686)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7346..8137,8138..8206))
                     /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     7969..7988
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8207..8311)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8386..8405)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8577..8877,1..79)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
       61 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      121 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      181 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      241 gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt
      301 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
      361 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
      421 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
      481 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
      541 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
      601 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
      661 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
      721 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
      781 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
      841 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
      901 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
      961 caaaagtaag accaccgcac agcaagcggc cggccgctga tcttcagacc tggaggagga
     1021 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1081 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1141 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1201 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1261 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1321 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1381 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1441 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1501 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1561 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1621 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1681 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1741 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     1801 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     1861 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     1921 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     1981 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2041 cagcagagat ccagtttggt tagtaccggg cccgctctag agatccgacg cgccatctct
     2101 aggcccgcgc cggccccctc gcacggactt gtgggagaag ctcggctact cccctgcccc
     2161 ggttaatttg catataatat ttcctagtaa ctatagaggc ttaatgtgcg ataaaagaca
     2221 gataatctgt tctttttaat actagctaca ttttacatga taggcttgga tttctataac
     2281 ttcgtatagc atacattata cgaagttata aacagcacaa aaggaaactc accctaactg
     2341 taaagtaatt gtgtgttttg agactataag tatcccttgg agaaccacct tgttggagga
     2401 tccactagtc cagtggttta agagctaagc tggaaacagc atagcaagtt taaataaggc
     2461 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttct cgagtactag
     2521 gatccattag gcggccgcgt ggataaccgt attaccgcca tgcatgtgcc cgtcagtggg
     2581 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
     2641 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
     2701 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     2761 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
     2821 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg
     2881 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
     2941 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
     3001 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
     3061 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     3121 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     3181 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     3241 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     3301 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     3361 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     3421 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     3481 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgcgc ttttggagta
     3541 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     3601 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     3661 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     3721 catttcaggt gtcgtgagct agcccaccat gaccgagtac aagcccacgg tgcgcctcgc
     3781 cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     3841 cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg agctgcaaga
     3901 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     3961 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     4021 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     4081 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     4141 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     4201 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     4261 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     4321 gtgcatgacc cgcaagcccg gtgccggatc gggagagggc agaggaagtc tgctaacatg
     4381 cggtgacgtc gaggagaatc ctggcccacc ggtcgccacc agcgagctga ttaaggagaa
     4441 catgcacatg aagctgtaca tggagggcac cgtggacaac catcacttca agtgcacatc
     4501 cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg
     4561 cggccctctc cccttcgcct tcgacatcct ggctactagc ttcctctacg gcagcaagac
     4621 cttcatcaac cacacccagg gcatccccga cttcttcaag cagtccttcc ctgagggctt
     4681 cacatgggag agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac
     4741 cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcacatc
     4801 caacggccct gtgatgcaga agaaaacact cggctgggag gccttcaccg agacgctgta
     4861 ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag
     4921 ccatctgatc gcaaacatca agaccacata tagatccaag aaacccgcta agaacctcaa
     4981 gatgcctggc gtctactatg tggactacag actggaaaga atcaaggagg ccaacaacga
     5041 gacctacgtc gagcagcacg aggtggcagt ggccagatac tgcgacctcc ctagcaaact
     5101 ggggcacaag cttaattaag aattcgtcga gggacctaat aacttcgtat agcatacatt
     5161 atacgaagtt atacatgttt aagggttccg gttccactag gtacaattcg atatcaagct
     5221 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     5281 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     5341 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     5401 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     5461 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     5521 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     5581 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     5641 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     5701 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     5761 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     5821 ataccgtcga cctcgatcga gacctagaaa aacatggagc aatcacaagt agcaatacag
     5881 cagctaccaa tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc
     5941 cagtcacacc tcaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     6001 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagata
     6061 tccttgatct gtggatctac cacacacaag gctacttccc tgattggcag aactacacac
     6121 cagggccagg gatcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg
     6181 agcaagagaa ggtagaagaa gccaatgaag gagagaacac ccgcttgtta caccctgtga
     6241 gcctgcatgg gatggatgac ccggagagag aagtattaga gtggaggttt gacagccgcc
     6301 tagcatttca tcacatggcc cgagagctgc atccggactg tactgggtct ctctggttag
     6361 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     6421 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     6481 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagcatg tgagcaaaag
     6541 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     6601 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6661 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6721 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6781 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6841 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6901 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6961 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     7021 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     7081 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     7141 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     7201 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     7261 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     7321 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     7381 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     7441 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     7501 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     7561 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     7621 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     7681 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7741 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7801 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7861 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7921 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7981 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     8041 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     8101 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     8161 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     8221 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     8281 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     8341 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     8401 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     8461 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     8521 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
     8581 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     8641 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     8701 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     8761 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     8821 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctg
//
