LOCUS       sequence-193736-        8360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA with two MCP binding sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8360)
  AUTHORS   Qin P, Parlak M, Kuscu C, Bandaria J, Mir M, Szlachta K, Singh R, 
            Darzacq X, Yildiz A, Adli M
  TITLE     Live cell imaging of low- and non-repetitive chromosome loci using 
            CRISPR-Cas9.
  JOURNAL   Nat Commun. 2017 Mar 14;8:14725. doi: 10.1038/ncomms14725.
  PUBMED    28290446
REFERENCE   2  (bases 1 to 8360)
  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: "Nat 
            Commun."; date: "2017-03-14"; volume: "8:14725. doi"; pages: " 
            10.1038/ncomms14725"
COMMENT     Sequence Label: sg2.02 MUC4.1
FEATURES             Location/Qualifiers
     source          1..8360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(195..228)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     232..252
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        359..377
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        434..452
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     enhancer        607..910
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        911..1114
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             1148..1744
                     /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(1148..1167)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1644..1664
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1754..1807
                     /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             1820..2521
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    complement(2541..2574)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2630..3218
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2683..2703)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3101..3112)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3221..3237)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3222..3238)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3747..3927
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3989..4577)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4069..4088)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4748..5539,5540..5608))
                     /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     5371..5390
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5609..5713)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5788..5807)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5979..6358
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6360..6558
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6576..6756
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6803..6928
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7425..7658
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7843..7887
                     /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    8185..8302
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 gatccgacgc gccatctcta ggcccgcgcc ggccccctcg cacggacttg tgggagaagc
       61 tcggctactc ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct
      121 taatgtgcga taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat
      181 aggcttggat ttctataact tcgtatagca tacattatac gaagttataa acagcacaaa
      241 aggaaactca ccctaactgt aaagtaattg tgtgttttga gactataagt atcccttgga
      301 gaaccacctt gttgggtaaa gtagaaaagg cataaagttt aagagctatg ctgggccaac
      361 atgaggatca cccatgtctg cagggcccag catagcaagt ttaaataagg ctagtccgtt
      421 atcaacttgg ccaacatgag gatcacccat gtctgcaggg ccaagtggca ccgagtcggt
      481 gctttttttc tcgagtacta ggatccatta ggcggccgcg tggataaccg tattaccgcc
      541 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      601 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      661 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      721 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      781 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      841 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      901 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      961 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     1021 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     1081 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
     1141 gcccaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtcccccg
     1201 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     1261 cccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     1321 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     1381 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     1441 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     1501 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     1561 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     1621 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     1681 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     1741 tgccggatcg ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc
     1801 tggcccaccg gtcgccacca tgagcgagct gattaaggag aacatgcaca tgaagctgta
     1861 catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc
     1921 ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc
     1981 cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca
     2041 gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac
     2101 cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg
     2161 cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca
     2221 gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct
     2281 ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat
     2341 caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta
     2401 tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca
     2461 cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatta
     2521 agaattcgtc gagggaccta ataacttcgt atagcataca ttatacgaag ttatacatgt
     2581 ttaagggttc cggttccact aggtacaatt cgatatcaag cttatcgata atcaacctct
     2641 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     2701 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     2761 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     2821 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     2881 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     2941 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     3001 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     3061 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     3121 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     3181 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgatc
     3241 gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc aatgctgatt
     3301 gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca cctcaggtac
     3361 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     3421 ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat ctgtggatct
     3481 accacacaca aggctacttc cctgattggc agaactacac accagggcca gggatcagat
     3541 atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag aaggtagaag
     3601 aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat gggatggatg
     3661 acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt catcacatgg
     3721 cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc tgagcctggg
     3781 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     3841 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     3901 tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa aaggccagga
     3961 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4021 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4081 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4141 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4201 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4261 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4321 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4381 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4441 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4501 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4561 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4621 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4681 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4741 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4801 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4861 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4921 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4981 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5041 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5101 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5161 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5221 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5281 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5341 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5401 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5461 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5521 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5581 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5641 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5701 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     5761 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5821 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     5881 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     5941 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta
     6001 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     6061 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     6121 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     6181 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     6241 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     6301 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     6361 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     6421 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     6481 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     6541 ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct
     6601 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     6661 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     6721 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     6781 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     6841 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     6901 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     6961 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     7021 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     7081 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     7141 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     7201 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     7261 gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     7321 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     7381 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     7441 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     7501 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     7561 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     7621 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     7681 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     7741 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     7801 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     7861 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     7921 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     7981 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     8041 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga
     8101 gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag
     8161 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
     8221 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     8281 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt
     8341 agtaccgggc ccgctctaga
//
