LOCUS       sequence-360244-        4972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression cloning vector for sgRNA-25xPBS9R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4972)
  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 4972)
  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: pAC11010-pCR8-sgRNA-25xPBS9R
FEATURES             Location/Qualifiers
     source          1..4972
                     /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(2851..2950)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2967..2986)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2968..2986)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2991..3007)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2991..3007)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3438..4229
                     /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      4322..4910
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4811..4830
                     /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 tataagattg ttgtatagcc
     2701 tgttgtatag cctgttgtat agcctgttgt atagcctgtt gtataagatt gttgtatagc
     2761 ctgttgtata gcctgttgta tagcctgttg tatagcctgt tgtataagat ctcgtattcg
     2821 tttttttggc gcgccttaat taagaattcg acccagcttt cttgtacaaa gttggcatta
     2881 taaaaaataa ttgctcatca atttgttgca acgaacaggt cactatcagt caaaataaaa
     2941 tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg gtcatagctg
     3001 tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa
     3061 atatatcatc atgcctcctc tagaccagcc aggacagaaa tgcctcgact tcgctgctac
     3121 ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc cagtggacat
     3181 aagcctgttc ggttcgtaag ctgtaatgca agtagcgtat gcgctcacgc aactggtcca
     3241 gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg
     3301 actgtttttt tggggtacag tctatgcctc gggcatccaa gcagcaagcg cgttacgccg
     3361 tgggtcgatg tttgatgtta tggagcagca acgatgttac gcagcagggc agtcgcccta
     3421 aaacaaagtt aaacattatg agggaagcgg tgatcgccga agtatcgact caactatcag
     3481 aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta catttgtacg
     3541 gctccgcagt ggatggcggc ctgaagccac acagtgatat tgatttgctg gttacggtga
     3601 ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg
     3661 cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt gtgcacgacg
     3721 acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa tggcagcgca
     3781 atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg gctatcttgc
     3841 tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag gaactctttg
     3901 atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg ctatggaact
     3961 cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt
     4021 acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg gcaatggagc
     4081 gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat cttggacaag
     4141 aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccac tacgtgaaag
     4201 gcgagatcac caaggtagtc ggcaaataac cctcgagcca cccatgacca aaatccctta
     4261 acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4321 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4381 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4441 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     4501 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4561 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4621 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4681 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4741 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4801 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4861 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4921 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg tt
//
