LOCUS       sequence-194883-        5890 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Basic cloning vector for the expression of PP7 inserted sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5890)
  AUTHORS   Shao S, Zhang W, Hu H, Xue B, Qin J, Sun C, Sun Y, Wei W, Sun Y
  TITLE     Long-term dual-color tracking of genomic loci by modified sgRNAs of 
            the CRISPR/Cas9 system.
  JOURNAL   Nucleic Acids Res. 2016 May 19;44(9):e86. doi: 10.1093/nar/gkw066. 
            Epub 2016 Feb 4.
  PUBMED    26850639
REFERENCE   2  (bases 1 to 5890)
  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: "Nucleic 
            Acids Res."; date: "2016-05-19"; volume: "44(9):e86. doi"; pages: " 
            10.1093/nar/gkw066. Epub 2016 Feb 4"
COMMENT     Sequence Label: psgRNA2.02-PP7-ccdB
FEATURES             Location/Qualifiers
     source          1..5890
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..270
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     22..42
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     193..212
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     294..313
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        294..312
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    342..466
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(895..1200)
                     /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(997..1016)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1520..2203)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2118..2137
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2204..2306)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2484..2608)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    2853..2974
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2864..2883)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2918..2937
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3222..3239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3393..3981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3473..3492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4152..4943,4944..5012))
                     /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     4775..4794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5013..5117)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5144..5599
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5231..5250)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5441..5462
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5730..5778
                     /note="synthetic polyadenylation signal"
     misc_feature    5792..5883
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5832..5851
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
       61 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      121 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      181 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      241 ttatatatct tgtggaaagg acgaaacacc gagagacgct ggcttatcga aattaatacg
      301 actcactata gggagaccca agctggctag ttaagctatc aacaagtttg tacaaaaaag
      361 ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
      421 aacagactac ataatactgt aaaacacaac atatccagtc actatgaatc aactacttag
      481 atggtattag tgacctgtag tcgaccgaca gccttccaaa tgttcttcgg gtgatgctgc
      541 caacttagtc gaccgacagc cttccaaatg ttcttctcaa acggaatcgt cgtatccagc
      601 ctactcgcta ttgtcctcaa tgccgtatta aatcataaaa agaaataaga aaaagaggtg
      661 cgagcctctt ttttgtgtga caaaataaaa acatctacct attcatatac gctagtgtca
      721 tagtcctgaa aatcatctgc atcaagaaca atttcacaac tcttatactt ttctcttaca
      781 agtcgttcgg cttcatctgg attttcagcc tctatactta ctaaacgtga taaagtttct
      841 gtaatttcta ctgtatcgac ctgcagactg gctgtgtata agggagcctg acatttatat
      901 tccccagaac atcaggttaa tggcgttttt gatgtcattt tcgcggtggc tgagatcagc
      961 cacttcttcc ccgataacgg agaccggcac actggccata tcggtggtca tcatgcgcca
     1021 gctttcatcc ccgatatgca ccaccgggta aagttcacgg gagactttat ctgacagcag
     1081 acgtgcactg gccaggggga tcaccatccg tcgcccgggc gtgtcaataa tatcactctg
     1141 tacatccaca aacagacgat aacggctctc tcttttatag gtgtaaacct taaactgcat
     1201 ttcaccagcc cctgttctcg tcagcaaaag agccgttcat ttcaataaac cgggcgacct
     1261 cagccatccc ttcctgattt tccgctttcc agcgttcggc acgcagacga cgggcttcat
     1321 tctgcatggt tgtgcttacc agaccggaga tattgacatc atatatgcct tgagcaactg
     1381 atagctgtcg ctgtcaactg tcactgtaat acgctgcttc atagcatacc tctttttgac
     1441 atacttcggg tatacatatc agtatatatt cttataccgc aaaaatcagc gcgcaaatac
     1501 gcatactgtt atctggcttt tagtaagccg gatccacgcg gcgtttacgc cccccctgcc
     1561 actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa
     1621 cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt
     1681 tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac
     1741 tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag
     1801 ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact
     1861 gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga
     1921 aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca
     1981 tacggaattc cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa
     2041 acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct
     2101 ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt
     2161 gggatatatc aacggtggta tatccagtga tttttttctc cattttagct tccttagctc
     2221 ctgaaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa
     2281 agttggaacc tcttacgtgc cgatcaacgt ctcattttcg ccaaaagttg gcccagggct
     2341 tcccggtatc aacagggaca ccaggattta tttattctgc gaagtgatct tccgtcacag
     2401 gtatttattc ggcgcaaagt gcgtcgggtg atgctgccaa cttagtcgac tacaggtcac
     2461 taataccatc taagtagttg attcatagtg actggatatg ttgtgtttta cagtattatg
     2521 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
     2581 ctcgttcagc tttcttgtac aaagtggttg atctagaggg cccgcggttc gaacgtctct
     2641 gtttaagagc tatgctgggc cgaaaggagc agacgatatg gcgtcgctcc ctgcggccca
     2701 gcatagcaag tttaaataag gctagtccgt tatcaacttg gccgaaagga gcagacgata
     2761 tggcgtcgct ccctgcggcc aagtggcacc gagtcggtgc tttttttttc gaggtcgacg
     2821 gtatagcggc cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac
     2881 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     2941 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     3001 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     3061 tggtaaaatc gataaggatc cgtcgaccga tgcccttgag agccttcaac ccagtcagct
     3121 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     3181 tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg
     3241 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3301 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3361 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3421 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3481 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3541 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3601 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3661 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3721 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3781 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3841 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3901 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3961 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4021 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4081 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4141 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4201 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4261 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4321 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4381 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4441 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4501 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4561 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4621 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4681 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4741 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4801 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4861 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4921 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4981 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5041 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5101 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc
     5161 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5221 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5281 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5341 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5401 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5461 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     5521 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5581 atattaacgc ttacaatttg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     5641 tcggtgcggg cctcttcgct attacgccag cccaagctac catgataagt aagtaatatt
     5701 aaggtacggg aggtacttgg agcggccgca ataaaatatc tttattttca ttacatctgt
     5761 gtgttggttt tttgtgtgaa tcgatagtac taacatacgc tctccatcaa aacaaaacga
     5821 aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag tgcaggtgcc agaacatttc
     5881 tctatcgata
//
