LOCUS       sequence-162537-        5334 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses minor satellite-specific sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5334)
  AUTHORS   Schmidtmann E, Anton T, Rombaut P, Herzog F, Leonhardt H
  TITLE     Determination of Local Chromatin Composition by CasID.
  JOURNAL   Nucleus. 2016 Sep 27:0.
  PUBMED    27676121
REFERENCE   2  (bases 1 to 5334)
  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: "Nucleus. 
            2016 Sep 27:0."
COMMENT     Sequence Label: pEX-A-U6-MiSgRNA_PuroR
FEATURES             Location/Qualifiers
     source          1..5334
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        70..318
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     70..90
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     241..260
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        340..415
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(427..451)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(486..508)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    497..513
                     /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(521..527,528..545,546..551))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    566..587
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(704..721)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(875..1463)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(955..974)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1634..2425,2426..2494))
                     /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     2257..2276
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2495..2599)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2667..2685
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2723..2745)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2845..2864
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2898..3397
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2933..2956)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3330..3349
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3421..4017
                     /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(3421..3440)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3917..3937
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4024..5019
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(5072..5089)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5078..5302
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 tgtacaaaaa agcaggcttt aaaggaacca attcagtcga ctggatccgg taccaaggtc
       61 gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
      121 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
      181 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
      241 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
      301 tggaaaggac gaaacaccga cactgaaaaa cacattcgtg ttttagagct agaaatagca
      361 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
      421 ttctagaccc agctttcttg tacaaagttg gcattaacct gcggccgcaa gcttggatcc
      481 gaattcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
      541 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
      601 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
      661 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
      721 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
      781 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
      841 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
      901 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
      961 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     1021 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     1081 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1141 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     1201 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     1261 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     1321 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     1381 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     1441 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     1501 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     1561 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     1621 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     1681 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     1741 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     1801 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     1861 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     1921 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     1981 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     2041 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     2101 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     2161 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     2221 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     2281 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     2341 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     2401 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     2461 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     2521 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     2581 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     2641 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     2701 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     2761 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     2821 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     2881 tgggtaccga gcttaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc
     2941 tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc
     3001 cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc
     3061 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg
     3121 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg
     3181 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc
     3241 tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc
     3301 gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc
     3361 cgcgctgttc tcctcttcct catctccggg cctttcgacc gatcatcaag cttgatcctc
     3421 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     3481 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     3541 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     3601 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     3661 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     3721 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     3781 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     3841 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     3901 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     3961 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgccgga
     4021 tccatgccca cgctactgcg ggtttatata gacggtcccc acgggatggg gaaaaccacc
     4081 accacgcaac tgctggtggc cctgggttcg cgcgacgata tcgtctacgt acccgagccg
     4141 atgacttact ggcgggtgct gggggcttcc gagacaatcg cgaacatcta caccacacaa
     4201 caccgcctcg accagggtga gatatcggcc ggggacgcgg cggtggtaat gacaagcgcc
     4261 cagataacaa tgggcatgcc ttatgccgtg accgacgccg ttctggctcc tcatatcggg
     4321 ggggaggctg ggagctcaca tgccccgccc ccggccctca ccctcatctt cgaccgccat
     4381 cccatcgccg ccctcctgtg ctacccggcc gcgcggtacc ttatgggcag catgaccccc
     4441 caggccgtgc tggcgttcgt ggccctcatc ccgccgacct tgcccggcac caacatcgtg
     4501 cttggggccc ttccggagga cagacacatc gaccgcctgg ccaaacgcca gcgccccggc
     4561 gagcggctgg acctggctat gctggctgcg attcgccgcg tttacgggct acttgccaat
     4621 acggtgcggt atctgcagtg cggcgggtcg tggcgggagg actggggaca gctttcgggg
     4681 acggccgtgc cgccccaggg tgccgagccc cagagcaacg cgggcccacg accccatatc
     4741 ggggacacgt tatttaccct gtttcgggcc cccgagttgc tggcccccaa cggcgacctg
     4801 tataacgtgt ttgcctgggc cttggacgtc ttggccaaac gcctccgttc catgcacgtc
     4861 tttatcctgg attacgacca atcgcccgcc ggctgccggg acgccctgct gcaacttacc
     4921 tccgggatgg tccagaccca cgtcaccacc cccggctcca taccgacgat atgcgacctg
     4981 gcgcgcacgt ttgcccggga gatgggggag gctaactgag ctctagagcg gccagtgtcg
     5041 cggtatcgat gatctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     5101 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     5161 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     5221 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     5281 ggggatgcgg tgggctctat ggcttctgag gcggaacagc tcgcggccgc aagc
//
