LOCUS       sequence-360239-        4831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression cloning vector for sgRNA-15xPBSc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4831)
  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 4831)
  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: pAC11006-pCR8-sgRNA-15xPBSc
FEATURES             Location/Qualifiers
     source          1..4831
                     /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(2710..2809)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2826..2845)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2827..2845)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2850..2866)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2850..2866)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3297..4088
                     /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      4181..4769
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4670..4689
                     /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 cagatttgat gtagccttga
     2521 tgtagccttg atgtagcctt gatgtagcct tgatgtaaga tttgatgtag ccttgatgta
     2581 gccttgatgt agccttgatg tagccttgat gtaagatttg atgtagcctt gatgtagcct
     2641 tgatgtagcc ttgatgtagc cttgatgtaa gatctttttt tgttttggcg cgccttaatt
     2701 aagaattcga cccagctttc ttgtacaaag ttggcattat aaaaaataat tgctcatcaa
     2761 tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc catccagctg
     2821 atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg
     2881 tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca tgcctcctct
     2941 agaccagcca ggacagaaat gcctcgactt cgctgctacc caaggttgcc gggtgacgca
     3001 caccgtggaa acggatgaag gcacgaaccc agtggacata agcctgttcg gttcgtaagc
     3061 tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg
     3121 gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt
     3181 ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat
     3241 ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacattatga
     3301 gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc
     3361 gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc
     3421 tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa
     3481 cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga
     3541 ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc
     3601 cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct
     3661 tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata
     3721 gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc
     3781 tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg
     3841 atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa
     3901 tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc
     3961 ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc
     4021 gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg
     4081 gcaaataacc ctcgagccac ccatgaccaa aatcccttaa cgtgagttac gcgtcgttcc
     4141 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4201 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4261 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4321 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4381 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4441 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4501 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4561 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4621 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4681 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4741 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4801 tggccttttg ctggcctttt gctcacatgt t
//
