LOCUS       sequence-182683-        5809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Basic cloning vector for the expression of imaging-based 
            optimization sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5809)
  AUTHORS   Shao S, Chang L, Sun Y, Hou Y, Fan X, Sun Y
  TITLE     Multiplexed sgRNA Expression Allows Versatile Single Nonrepetitive 
            DNA Labeling and Endogenous Gene Regulation.
  JOURNAL   ACS Synth Biol. 2017 Sep 7. doi: 10.1021/acssynbio.7b00268.
  PUBMED    28849913
REFERENCE   2  (bases 1 to 5809)
  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: "ACS Synth 
            Biol. 2017 Sep 7. doi: 10.1021/acssynbio.7b00268."
COMMENT     Sequence Label: psgRNA-ccdB
FEATURES             Location/Qualifiers
     source          1..5809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    123..171
                     /note="synthetic polyadenylation signal"
     misc_feature    185..276
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     225..244
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        305..552
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     475..494
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     576..595
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        576..594
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    624..748
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1177..1482)
                     /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(1279..1298)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1802..2485)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2400..2419
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2486..2588)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2766..2890)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3055..3176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3066..3085)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3120..3139
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3424..3441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3595..4183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3675..3694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4354..5145,5146..5214))
                     /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     4977..4996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5215..5319)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5346..5801
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5433..5452)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5643..5664
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
       61 cagcccaagc taccatgata agtaagtaat attaaggtac gggaggtact tggagcggcc
      121 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
      181 tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca aaataggctg
      241 tccccagtgc aagtgcaggt gccagaacat ttctctatcg ataggtacca aggtcgggca
      301 ggaagagggc ctatttccat gattccttca tatttgcata tacgatacaa ggctgttaga
      361 gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
      421 agaaagtaat aatttcttgg gtatttgcag tttttaaaat tatgttttaa aatggactat
      481 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
      541 ggacgaaaca ccgagagacg ctggcttatc gaaattaata cgactcacta tagggagacc
      601 caagctggct agttaagcta tcaacaagtt tgtacaaaaa agctgaacga gaaacgtaaa
      661 atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact
      721 gtaaaacaca acatatccag tcactatgaa tcaactactt agatggtatt agtgacctgt
      781 agtcgaccga cagccttcca aatgttcttc gggtgatgct gccaacttag tcgaccgaca
      841 gccttccaaa tgttcttctc aaacggaatc gtcgtatcca gcctactcgc tattgtcctc
      901 aatgccgtat taaatcataa aaagaaataa gaaaaagagg tgcgagcctc ttttttgtgt
      961 gacaaaataa aaacatctac ctattcatat acgctagtgt catagtcctg aaaatcatct
     1021 gcatcaagaa caatttcaca actcttatac ttttctctta caagtcgttc ggcttcatct
     1081 ggattttcag cctctatact tactaaacgt gataaagttt ctgtaatttc tactgtatcg
     1141 acctgcagac tggctgtgta taagggagcc tgacatttat attccccaga acatcaggtt
     1201 aatggcgttt ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac
     1261 ggagaccggc acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg
     1321 caccaccggg taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg
     1381 gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc tgtacatcca caaacagacg
     1441 ataacggctc tctcttttat aggtgtaaac cttaaactgc atttcaccag cccctgttct
     1501 cgtcagcaaa agagccgttc atttcaataa accgggcgac ctcagccatc ccttcctgat
     1561 tttccgcttt ccagcgttcg gcacgcagac gacgggcttc attctgcatg gttgtgctta
     1621 ccagaccgga gatattgaca tcatatatgc cttgagcaac tgatagctgt cgctgtcaac
     1681 tgtcactgta atacgctgct tcatagcata cctctttttg acatacttcg ggtatacata
     1741 tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat acgcatactg ttatctggct
     1801 tttagtaagc cggatccacg cggcgtttac gccccccctg ccactcatcg cagtactgtt
     1861 gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa
     1921 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg
     1981 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg
     2041 gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt
     2101 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt
     2161 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt
     2221 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag
     2281 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
     2341 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag
     2401 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg
     2461 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact
     2521 caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt
     2581 gccgatcaac gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga
     2641 caccaggatt tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa
     2701 gtgcgtcggg tgatgctgcc aacttagtcg actacaggtc actaatacca tctaagtagt
     2761 tgattcatag tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca
     2821 aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca gctttcttgt
     2881 acaaagtggt tgatctagag ggcccgcggt tcgaacgtct ctgtttaaga gctatgctgg
     2941 aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     3001 gtcggtgctt ttttttctag agtcggggcg gccggccgct tcgagcagac atgataagat
     3061 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     3121 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     3181 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     3241 gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc gatgcccttg
     3301 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
     3361 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc
     3421 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3481 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3541 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3601 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3661 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3721 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3781 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3841 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3901 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3961 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4021 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4081 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4141 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4201 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4261 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4321 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4381 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4441 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4501 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4561 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4621 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4681 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4741 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4801 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4861 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4921 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4981 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5041 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5101 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5161 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5221 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5281 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5341 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     5401 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     5461 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     5521 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     5581 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     5641 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     5701 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     5761 atttaacgcg aattttaaca aaatattaac gtttacaatt tcccattcg
//
