LOCUS       sequence-194882-        5899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Basic cloning vector for the expression of MS2 inserted sgRNA .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5899)
  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 5899)
  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-MS2-ccdB
FEATURES             Location/Qualifiers
     source          1..5899
                     /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"
     misc_RNA        2663..2681
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2738..2756
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     polyA_signal    2862..2983
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2873..2892)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2927..2946
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3231..3248)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3402..3990)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3482..3501)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4161..4952,4953..5021))
                     /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     4784..4803
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5022..5126)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5153..5608
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5240..5259)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5450..5471
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5739..5787
                     /note="synthetic polyadenylation signal"
     misc_feature    5801..5892
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5841..5860
                     /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 caacatgagg atcacccatg tctgcagggc ccagcatagc
     2701 aagtttaaat aaggctagtc cgttatcaac ttggccaaca tgaggatcac ccatgtctgc
     2761 agggccaagt ggcaccgagt cggtgctttt ttttctagac ccagctttct gcattactcg
     2821 aggtcgacgg tatggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt
     2881 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     2941 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     3001 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     3061 ctacaaatgt ggtaaaatcg ataaggatcc gtcgaccgat gcccttgaga gccttcaacc
     3121 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
     3181 tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc ctcgctcact
     3241 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3301 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3361 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3421 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3481 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3541 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3601 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3661 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3721 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3781 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3841 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3901 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3961 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4021 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4081 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4141 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4201 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4261 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4321 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4381 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4441 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4501 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4561 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4621 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4681 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4741 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4801 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4861 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4921 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4981 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5041 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5101 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc
     5161 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5221 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5281 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5341 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     5401 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5461 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     5521 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5581 tttaacaaaa tattaacgct tacaatttgc cattcgccat tcaggctgcg caactgttgg
     5641 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc ccaagctacc atgataagta
     5701 agtaatatta aggtacggga ggtacttgga gcggccgcaa taaaatatct ttattttcat
     5761 tacatctgtg tgttggtttt ttgtgtgaat cgatagtact aacatacgct ctccatcaaa
     5821 acaaaacgaa acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagt gcaggtgcca
     5881 gaacatttct ctatcgata
//
