LOCUS       sequence-334832-        7578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  backbone for sgRNA expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7578)
  AUTHORS   Wang H, Xu X, Nguyen CM, Liu Y, Gao Y, Lin X, Daley T, Kipniss NH, 
            La Russa M, Qi LS
  TITLE     CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear 
            Organization.
  JOURNAL   Cell. 2018 Nov 15;175(5):1405-1417.e14. doi: 
            10.1016/j.cell.2018.09.013. Epub 2018 Oct 11.
  PUBMED    30318144
REFERENCE   2  (bases 1 to 7578)
  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.2018.09.013"; journalName: "Cell"; date: 
            "2018-11-15- 15"; volume: "175"; issue: "5"; pages: "1405-1417.e14"
COMMENT     Sequence Label: pHR-U6-sgRNA-CMV-puro
FEATURES             Location/Qualifiers
     source          1..7578
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..240
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             274..870
                     /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(274..293)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     770..790
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             880..933
                     /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"
     protein_bind    complement(956..989)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1045..1633
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1098..1118)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1516..1527)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1636..1652)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1637..1653)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             2162..2342
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(2404..2992)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2484..2503)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3163..3954,3955..4023))
                     /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     3786..3805
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4024..4128)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4203..4222)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4394..4773
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4775..4973
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             4991..5171
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5218..5343
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5840..6073
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6258..6302
                     /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    6600..6717
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(6970..7003)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     7007..7027
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        7111..7186
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        join(7311..7578,1..36)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
       61 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      121 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      181 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      241 ggtttagtga accgtcagat ccgctagccc accatgaccg agtacaagcc cacggtgcgc
      301 ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac
      361 taccccgcca cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg
      421 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
      481 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
      541 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
      601 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
      661 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
      721 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
      781 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
      841 acctggtgca tgacccgcaa gcccggtgcc ggatcgggag agggcagagg aagtctgcta
      901 acatgcggtg acgtcgagga gaatcctggc ccaccggaat tcgtcgaggg acctaataac
      961 ttcgtatagc atacattata cgaagttata catgtttaag ggttccggtt ccactaggta
     1021 caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     1081 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     1141 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     1201 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     1261 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     1321 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     1381 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     1441 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     1501 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     1561 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     1621 gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac atggagcaat
     1681 cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga
     1741 ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc
     1801 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     1861 ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga
     1921 ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta
     1981 caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag agaacacccg
     2041 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tattagagtg
     2101 gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc cggactgtac
     2161 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     2221 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     2281 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     2341 cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2401 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2461 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2521 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2581 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2641 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2701 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2761 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2821 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     2881 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2941 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3001 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3061 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3121 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3181 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3241 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3301 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3361 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3421 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3481 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3541 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3601 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3661 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3721 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3781 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     3841 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     3901 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     3961 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4021 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4081 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4141 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     4201 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     4261 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     4321 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     4381 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     4441 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     4501 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     4561 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     4621 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     4681 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     4741 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     4801 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     4861 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     4921 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagcgcgttt
     4981 tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     5041 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     5101 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     5161 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
     5221 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
     5281 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
     5341 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
     5401 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
     5461 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     5521 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     5581 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     5641 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg ctgatcttca
     5701 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     5761 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     5821 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     5881 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     5941 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     6001 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     6061 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     6121 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     6181 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     6241 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     6301 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     6361 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     6421 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca
     6481 ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag
     6541 tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt catccacaat
     6601 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
     6661 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
     6721 gtttattaca gggacagcag agatccagtt tggttagtac cgggcccgct ctagagatcc
     6781 gacgcgccat ctctaggccc gcgccggccc cctcgcacgg acttgtggga gaagctcggc
     6841 tactcccctg ccccggttaa tttgcatata atatttccta gtaactatag aggcttaatg
     6901 tgcgataaaa gacagataat ctgttctttt taatactagc tacattttac atgataggct
     6961 tggatttcta taacttcgta tagcatacat tatacgaagt tataaacagc acaaaaggaa
     7021 actcacccta actgtaaagt aattgtgtgt tttgagacta taagtatccc ttggagaacc
     7081 accttgttgg aacgactagt taggcgtgta gttttagagc tagaaatagc aagttaaaat
     7141 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttctcgagt
     7201 actaggatcc attaggcggc cgcgtggata accgtattac cgccatgcat tagttattaa
     7261 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     7321 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     7381 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     7441 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     7501 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     7561 tgggactttc ctacttgg
//
