LOCUS       Exported                5898 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Basic cloning vector for the expression of MS2 inserted sgRNA .
ACCESSION   .
VERSION     .
KEYWORDS    psgRNA2.02-MS2-ccdB
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5898)
  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 5898)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5898
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        22..261
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     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(1549..2202)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG"
     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"
     misc_RNA        2662..2680
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2737..2755
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     polyA_signal    2861..2982
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2872..2891)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2926..2945
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3230..3247)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3401..3989)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3481..3500)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4160..5020)
                     /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     4783..4802
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5021..5125)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5152..5607
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5239..5258)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5449..5470
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5738..5786
                     /note="synthetic polyadenylation signal"
     misc_feature    5800..5891
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5840..5859
                     /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 aacatgagga tcacccatgt ctgcagggcc cagcatagca
     2701 agtttaaata aggctagtcc gttatcaact tggccaacat gaggatcacc catgtctgca
     2761 gggccaagtg gcaccgagtc ggtgcttttt tttctagacc cagctttctg cattactcga
     2821 ggtcgacggt atggcggccg gccgcttcga gcagacatga taagatacat tgatgagttt
     2881 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     2941 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     3001 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     3061 tacaaatgtg gtaaaatcga taaggatccg tcgaccgatg cccttgagag ccttcaaccc
     3121 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     3181 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg
     3241 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3301 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3361 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3421 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3481 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3541 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     3601 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3661 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3721 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3781 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3841 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3901 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3961 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4021 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4081 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4141 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4201 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4261 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4321 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4381 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4441 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4501 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4561 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4621 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4681 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4741 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4801 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4861 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4921 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4981 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5041 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5101 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct
     5161 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5221 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5281 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5341 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     5401 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5461 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     5521 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5581 ttaacaaaat attaacgttt acaatttccc attcgccatt caggctgcgc aactgttggg
     5641 aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc caagctacca tgataagtaa
     5701 gtaatattaa ggtacgggag gtacttggag cggccgcaat aaaatatctt tattttcatt
     5761 acatctgtgt gttggttttt tgtgtgaatc gatagtacta acatacgctc tccatcaaaa
     5821 caaaacgaaa caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg caggtgccag
     5881 aacatttctc tatcgata
//
