LOCUS       sequence-182680-        5889 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 5889)
  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 5889)
  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..5889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..269
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     192..211
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     293..312
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        293..311
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    341..465
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(894..1199)
                     /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(996..1015)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1519..2202)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2117..2136
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2203..2305)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2483..2607)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    2852..2973
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2863..2882)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2917..2936
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3221..3238)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3392..3980)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3472..3491)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4151..4942,4943..5011))
                     /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     4774..4793
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5012..5116)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5143..5598
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5230..5249)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5440..5461
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5729..5777
                     /note="synthetic polyadenylation signal"
     misc_feature    5791..5882
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5831..5850
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccaagg tcgggcagga agagggccta tttccatgat tccttcatat ttgcatatac
       61 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
      121 tacaaaatac gtgacgtaga aagtaataat ttcttgggta tttgcagttt ttaaaattat
      181 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
      241 tatatatctt gtggaaagga cgaaacaccg agagacgctg gcttatcgaa attaatacga
      301 ctcactatag ggagacccaa gctggctagt taagctatca acaagtttgt acaaaaaagc
      361 tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa
      421 acagactaca taatactgta aaacacaaca tatccagtca ctatgaatca actacttaga
      481 tggtattagt gacctgtagt cgaccgacag ccttccaaat gttcttcggg tgatgctgcc
      541 aacttagtcg accgacagcc ttccaaatgt tcttctcaaa cggaatcgtc gtatccagcc
      601 tactcgctat tgtcctcaat gccgtattaa atcataaaaa gaaataagaa aaagaggtgc
      661 gagcctcttt tttgtgtgac aaaataaaaa catctaccta ttcatatacg ctagtgtcat
      721 agtcctgaaa atcatctgca tcaagaacaa tttcacaact cttatacttt tctcttacaa
      781 gtcgttcggc ttcatctgga ttttcagcct ctatacttac taaacgtgat aaagtttctg
      841 taatttctac tgtatcgacc tgcagactgg ctgtgtataa gggagcctga catttatatt
      901 ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct gagatcagcc
      961 acttcttccc cgataacgga gaccggcaca ctggccatat cggtggtcat catgcgccag
     1021 ctttcatccc cgatatgcac caccgggtaa agttcacggg agactttatc tgacagcaga
     1081 cgtgcactgg ccagggggat caccatccgt cgcccgggcg tgtcaataat atcactctgt
     1141 acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt aaactgcatt
     1201 tcaccagccc ctgttctcgt cagcaaaaga gccgttcatt tcaataaacc gggcgacctc
     1261 agccatccct tcctgatttt ccgctttcca gcgttcggca cgcagacgac gggcttcatt
     1321 ctgcatggtt gtgcttacca gaccggagat attgacatca tatatgcctt gagcaactga
     1381 tagctgtcgc tgtcaactgt cactgtaata cgctgcttca tagcatacct ctttttgaca
     1441 tacttcgggt atacatatca gtatatattc ttataccgca aaaatcagcg cgcaaatacg
     1501 catactgtta tctggctttt agtaagccgg atccacgcgg cgtttacgcc ccccctgcca
     1561 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacaaac
     1621 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt
     1681 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact
     1741 ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg
     1801 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg
     1861 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa
     1921 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat
     1981 acggaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa
     2041 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg
     2101 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg
     2161 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc
     2221 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa
     2281 gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt
     2341 cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg
     2401 tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac ttagtcgact acaggtcact
     2461 aataccatct aagtagttga ttcatagtga ctggatatgt tgtgttttac agtattatgt
     2521 agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc
     2581 tcgttcagct ttcttgtaca aagtggttga tctagagggc ccgcggttcg aacgtctctg
     2641 tttaagagct atgctgggcc gaaaggagca gacgatatgg cgtcgctccc tgcggcccag
     2701 catagcaagt ttaaataagg ctagtccgtt atcaacttgg ccgaaaggag cagacgatat
     2761 ggcgtcgctc cctgcggcca agtggcaccg agtcggtgct ttttttttcg aggtcgacgg
     2821 tatggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc
     2881 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     2941 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     3001 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     3061 ggtaaaatcg ataaggatcc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc
     3121 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     3181 gcaactcgta ggacaggtgc cggcagcgct cttccgcttc ctcgctcact gactcgctgc
     3241 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3301 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3361 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3421 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3481 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3541 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
     3601 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3661 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3721 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3781 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     3841 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3901 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3961 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4021 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4081 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4141 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4201 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4261 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4321 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4381 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4441 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4501 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4561 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4621 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4681 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4741 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4801 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4861 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4921 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4981 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5041 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5101 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg
     5161 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     5221 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5281 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5341 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5401 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5461 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5521 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5581 tattaacgtt tacaatttcc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     5641 cggtgcgggc ctcttcgcta ttacgccagc ccaagctacc atgataagta agtaatatta
     5701 aggtacggga ggtacttgga gcggccgcaa taaaatatct ttattttcat tacatctgtg
     5761 tgttggtttt ttgtgtgaat cgatagtact aacatacgct ctccatcaaa acaaaacgaa
     5821 acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagt gcaggtgcca gaacatttct
     5881 ctatcgata
//
