LOCUS       sequence-207589-        4910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty guide RNA cloning vector with an AflII site added from Addgene
            41824.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4910)
  AUTHORS   Carleton JB, Berrett KC, Gertz J
  TITLE     Multiplex Enhancer Interference Reveals Collaborative Control of 
            Gene Regulation by Estrogen Receptor alpha-Bound Enhancers.
  JOURNAL   Cell Syst. 2017 Oct 25;5(4):333-344.e5. doi: 
            10.1016/j.cels.2017.08.011. Epub 2017 Sep 27.
  PUBMED    28964699
REFERENCE   2  (bases 1 to 4910)
  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.cels.2017.08.011"; journalName: "Cell Syst"; date: 
            "2017-10-25- 25"; volume: "5"; issue: "4"; pages: "333-344.e5"
COMMENT     Sequence Label: pGL3-U6-sgRNA-PGK-puromycin-AflII
FEATURES             Location/Qualifiers
     source          1..4910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    136..257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(147..166)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     201..220
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(505..522)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(676..1264)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(756..775)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1435..2226,2227..2295))
                     /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     2058..2077
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2296..2400)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2427..2882
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2514..2533)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2724..2745
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    3013..3061
                     /note="synthetic polyadenylation signal"
     misc_feature    3075..3166
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     3115..3134
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     3245..3265
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3416..3435
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(3542..3566)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3602..3719
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3768..4278
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4197..4215
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4300..4899
                     /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(4300..4319)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4796..4816
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 cgacccgcag cgcccgaccg aaaggagcgc acgaccccat gcatcggtac ctttaagacc
       61 aatgacttac aaggcagctg tagatcttag ccactttcta gagtcggggc ggccggccgc
      121 ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
      181 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
      241 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
      301 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg
      361 atccgtcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
      421 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
      481 gtgccggcag cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
      541 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      601 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      661 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      721 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      781 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      841 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      901 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      961 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     1021 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     1081 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     1141 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1201 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1261 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1321 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     1381 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     1441 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1501 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1561 tgcaatgata ccgcgggacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1621 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1681 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1741 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1801 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1861 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1921 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1981 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     2041 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     2101 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     2161 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     2221 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     2281 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     2341 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     2401 cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg
     2461 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2521 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2581 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2641 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2701 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2761 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2821 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2881 ttgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     2941 gctattacgc cagcccaagc taccatgata agtaagtaat attaaggtac gggaggtact
     3001 tggagcggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt
     3061 gaatcgatag tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca
     3121 aaataggctg tccccagtgc aagtgcaggt gccagaacat ttctctatcg ataggtaccg
     3181 attagtgaac ggatctcgac ggtatcgatc acgagactag cctcgagcgg ccgccccctt
     3241 caccgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     3301 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     3361 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     3421 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttaagtta
     3481 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttcta
     3541 gacccagctt tcttgtacaa agttgctcga cctcgagaca aatggcagta ttcatccaca
     3601 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     3661 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
     3721 gggtttatta cagggacagc agagatccac tttggccgcg gctcgagggg gttggggttg
     3781 cgccttttcc aaggcagccc tgggtttgcg cagggacgcg gctgctctgg gcgtggttcc
     3841 gggaaacgca gcggcgccga ccctgggact cgcacattct tcacgtccgt tcgcagcgtc
     3901 acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc tccgccccta
     3961 agtcgggaag gttccttgcg gttcgcggcg tgccggacgt gacaaacgga agccgcacgt
     4021 ctcactagta ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg ccgaccgcga
     4081 tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg ccgggaaggg
     4141 gcggtgcggg aggcggggtg tggggcggta gtgtgggccc tgttcctgcc cgcgcggtgt
     4201 tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt tgaccgaatc
     4261 accgacctct ctccccaggg ggatccaccg gagcttacca tgaccgagta caagcccacg
     4321 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     4381 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     4441 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     4501 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     4561 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     4621 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     4681 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     4741 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg aaacctccgc gccccgcaac
     4801 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     4861 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca
//
