LOCUS       sequence-360247-        4850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression cloning vector for sgRNA-15xPBS9R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4850)
  AUTHORS   Clow PA, Du M, Jillette N, Taghbalout A, Zhu JJ, Cheng AW
  TITLE     CRISPR-mediated multiplexed live cell imaging of nonrepetitive 
            genomic loci with one guide RNA per locus.
  JOURNAL   Nat Commun. 2022 Apr 6;13(1):1871. doi: 10.1038/s41467-022-29343-z.
  PUBMED    35387989
REFERENCE   2  (bases 1 to 4850)
  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: "2022-04-6"; volume: "13(1):1871. doi"; pages: " 
            10.1038/s41467-022-29343-z"
COMMENT     Sequence Label: pAC11007-pCR8-sgRNA-15xPBS9R
FEATURES             Location/Qualifiers
     source          1..4850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        683..931
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     683..703
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     854..873
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(980..1285)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1082..1101)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1627..2286)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     NNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2201..2220
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2340..2346,2347..2364,2365..2370))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2729..2828)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2845..2864)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2846..2864)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2869..2885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2869..2885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3316..4107
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      4200..4788
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4689..4708
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgatca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtattcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gaattcaccg gtgagggcct atttcccatg attccttcat atttgcatat
      721 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      781 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      841 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      901 tttatatatc ttgtggaaag gacgaaacac cgggtcttcg tcgacctgca gactggctgt
      961 gtataaggga gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt
     1021 cattttcgcg gtggctgaga tcagccactt cttccccgat aacggacacc ggcacactgg
     1081 ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt
     1141 cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc
     1201 cgggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt
     1261 tataggtgta aaccttaaac tgcatttcac cagcccctgt tctcgtcagc aaaagagccg
     1321 ttcatttcaa taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt
     1381 tcggcacgca gacgacgggc ttcattctgc atggttgtgc ttaccagacc ggagatattg
     1441 acatcatata tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct
     1501 gcttcatagc atacctcttt tttacatact tcgggtatac atatcagtat atattcttat
     1561 accgcaaaaa tcagcgcgca aatacgcata ctgttatctg gcttttagta agccggatcc
     1621 agatctttac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct
     1681 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
     1741 cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgttcat
     1801 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
     1861 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
     1921 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga
     1981 aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
     2041 cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag
     2101 aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
     2161 cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
     2221 aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
     2281 ctccatttta gcttccttag ctcctgaaaa tctcgacgga tcctaactca aaatccacac
     2341 attatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgcgg ccgcgaagac
     2401 ctgtttaaga gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca
     2461 acttgaaaaa gtggcaccga gtcggtgcca attgggtctc cagattgttg tatagcctgt
     2521 tgtatagcct gttgtatagc ctgttgtata gcctgttgta taagattgtt gtatagcctg
     2581 ttgtatagcc tgttgtatag cctgttgtat agcctgttgt ataagattgt tgtatagcct
     2641 gttgtatagc ctgttgtata gcctgttgta tagcctgttg tataagatct cgtattcgtt
     2701 tttttggcgc gccttaatta agaattcgac ccagctttct tgtacaaagt tggcattata
     2761 aaaaataatt gctcatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc
     2821 attatttgcc atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt
     2881 tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat
     2941 atatcatcat gcctcctcta gaccagccag gacagaaatg cctcgacttc gctgctaccc
     3001 aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca gtggacataa
     3061 gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga
     3121 accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac
     3181 tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
     3241 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
     3301 acaaagttaa acattatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
     3361 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
     3421 tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc
     3481 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
     3541 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
     3601 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
     3661 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
     3721 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
     3781 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
     3841 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     3901 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     3961 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     4021 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc
     4081 gagatcacca aggtagtcgg caaataaccc tcgagccacc catgaccaaa atcccttaac
     4141 gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4201 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4261 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4321 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     4381 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4441 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4501 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4561 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4621 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4681 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4741 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4801 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
//
