LOCUS       sequence-360252-        4887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression cloning vector for sgRNA-20xPBSc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4887)
  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 4887)
  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: pAC11009-pCR8-sgRNA-20xPBSc
FEATURES             Location/Qualifiers
     source          1..4887
                     /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(2766..2865)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2882..2901)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2883..2901)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2906..2922)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2906..2922)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3353..4144
                     /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      4237..4825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4726..4745
                     /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 gatttgatgt agccttgatg tagccttgat
     2701 gtagccttga tgtagccttg atgtaagatc tttttttgtt ttggcgcgcc ttaattaaga
     2761 attcgaccca gctttcttgt acaaagttgg cattataaaa aataattgct catcaatttg
     2821 ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgatat
     2881 cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc
     2941 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgcc tcctctagac
     3001 cagccaggac agaaatgcct cgacttcgct gctacccaag gttgccgggt gacgcacacc
     3061 gtggaaacgg atgaaggcac gaacccagtg gacataagcc tgttcggttc gtaagctgta
     3121 atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg
     3181 taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgggg tacagtctat
     3241 gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag
     3301 cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca ttatgaggga
     3361 agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca
     3421 tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa
     3481 gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg
     3541 gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct
     3601 ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc
     3661 taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga
     3721 gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt
     3781 tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt
     3841 tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga
     3901 gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc
     3961 gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt
     4021 catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc
     4081 agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa
     4141 ataaccctcg agccacccat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     4201 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4261 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4321 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4381 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4441 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4501 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4561 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4621 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4681 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4741 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4801 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4861 cttttgctgg ccttttgctc acatgtt
//
