LOCUS       sequence-212460-        7418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA entry plasmid for cloning guides - contains stuffer with BsmBI 
            sites - integrates at RPS1 (Part of Duet system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7418)
  AUTHORS   Vyas VK, Barrasa MI, Fink GR
  TITLE     A CRISPR system permits genetic engineering of essential genes and 
            gene families.
  JOURNAL   Sci Adv. 2015;1(3):e1500248.
  PUBMED    25977940
REFERENCE   2  (bases 1 to 7418)
  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: "Sci Adv."; 
            date: "2015"; volume: "1(3)"; pages: "e1500248"
COMMENT     Sequence Label: pV1090
FEATURES             Location/Qualifiers
     source          1..7418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      6..461
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(93..112)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     303..324
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        487..591
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(592..660,661..1452)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     KLDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(810..829)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1623..2211
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2112..2131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2365..2382
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2499..2520
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2535..2540,2541..2558,2559..2565)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2573..2589
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2578..2600
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2597..2613
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2597..2613
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2632..2652
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2634..2652
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_RNA        6114..6189
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(7237..7256)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7238..7256)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7266..7283)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7266..7282)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7275..7297)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
       61 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
      121 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
      181 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
      241 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
      301 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
      361 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
      421 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
      481 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
      541 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
      601 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
      661 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
      721 tacatcaaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
      781 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
      841 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
      901 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
      961 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     1021 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     1081 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     1141 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     1201 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     1261 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     1321 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     1381 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     1441 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     1501 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     1561 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1621 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     1681 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     1741 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     1801 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     1861 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     1921 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     1981 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2041 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2101 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2161 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2221 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2281 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     2341 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     2401 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     2461 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     2521 taggcacccc aggctttaca ctttatgctc ccggctcgta tgttgtgtgg aattgtgagc
     2581 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
     2641 cctcactaaa gggaacaaaa gctgggtacc tggttgttaa gtcagtagat gatttgttgt
     2701 tgtcgtttga ttttgttaca gcgtaaccag tgcgttttgt ttgtttccac atcatacact
     2761 tcactgaaac taaataagtt tgtttacatt ttgagacttc aggtacgacc cagggttgcg
     2821 acaaagttta ggtagtttgt cgtctgaatg tcgcaacaaa atagggctgt agccctagtc
     2881 atgtgatgtg aattaacaga acaagaagaa ctgctggtgc gcaaaaagat tatgtgtatt
     2941 ttatgtgcgt tgttatcctg cacactaaaa ttgagcagtg tacacacaca catcttgggc
     3001 tgtattttta ttcttgtttt tctggtgttc tctcactgtt aagctctaag tgaatttgtg
     3061 tgtgctgtaa tagtgtgtgt gttccaagtc ccagctctca cagatactca cgcacgccca
     3121 tactactgaa aatttcctga ctttctgtat ctaaaaattt tttactagga atttttttct
     3181 tttacgtttt tcacttgttt catataatca ccaactcaag tacaacagat ctggaccacc
     3241 tttgattgta aatagtaata attaccaccc ttatctaatt atttatttaa cttatttatt
     3301 tatttattat acatatatac aaatctaata aagtgaaaat ctcccccttc acacttcaca
     3361 tatgttaggc gtcatcctgt gctcccgaga accagtacca gtacatcgct gtttcgttcg
     3421 agacttgagg tctagtttta tacgtgaaga ggtcaatgcc gccgagagta aagccacatt
     3481 ttgcgtacaa attgcaggca ggtacattgt tcgtttgtgt ctctaatcgt atgccaagga
     3541 gctgtctgct tagtgcccac tttttcgcaa attcgatgag actgtgcgcg actcctttgc
     3601 ctcggtgcgt gtgcgacaca acaatgtgtt cgatagaggc tagatcgttc catgttgagt
     3661 tgagttcaat cttcccgaca agctcttggt cgatgaatgc gccatagcaa gcagagtctt
     3721 catcagagtc atcatctgag atgtaatcct tccggtaggg gctcacactt ctggtagata
     3781 gttcaaagcc ttggtcggat aggtgcacat cgaacacttc acgaacaatg aaatggttct
     3841 cagcatccaa tgtttccgcc acctgctcag ggatcaccga aattttcata tgagaaccgt
     3901 tatcgataac taaagcagca acttcttcta taaaaatggg ttagtatgac agtcatttaa
     3961 ataaggaatt tttcagttgg cttggtttca attcaatgtt cgtttttttt tttcttgctg
     4021 tgtttgtgtt tgtgttgttt atagttgtgt gcactgatcg tcgaaaaaaa aaattcatag
     4081 tgagccggga aatctgtata gcccagataa caacacaagt ccaaactaga aactcgtcaa
     4141 acaccaaaag caatgttgaa tcaattgcct tgcacaagta cacgtaggaa aacataaaac
     4201 attgcaattt tgaatattga gccttttgtc gtaacattga ttgataggat tactcaccga
     4261 atggttttga aaccactgcc gacagatcaa tcaatcaatc aaaaaacgtg aactttgaaa
     4321 aaggggaaga acagatacat tgaagttagc catttccact gatcgtcaca acatatctga
     4381 taaattactt tcaaaattat aagctgatgt gtgtgtatta ttaatgtgac agtaacatcc
     4441 caaacgagaa atattatctc gacaacaaaa aagtttgatc tgaattgaaa atgaagtttt
     4501 cccaccctac ccatttgtca tattgaaacc aatcaactga ttaatcaatc aattagaatt
     4561 gaagctaaac taaaacatac caccgtccat tttgaatgat tatatttttt taatattaat
     4621 atcgagataa tgtttctaag aaagaaagaa aaccaggagt gaaaattaga aaaggaaagg
     4681 aaagaaaaaa agaaaaatct gaaaatatat aaaaaaaaat tgtttcgttg gcaataaatc
     4741 ttggtgagaa cagcgaccga aagcaaataa gaacaaaata tgagtgtatt acgttgaaca
     4801 actaattaac gtgtgtgtat ggatcttttt ttcttttttc tctttaaccg actataaaca
     4861 acaaacattt ttgggcagtg cacacactac ttaatataca cagcataaat tacacgatta
     4921 gaaacaaatt agcttattaa aataacctaa tcaaaccgaa tattttatgg tattatgagt
     4981 aaactatata atataaatag cacacaccca caacaacaac aaaggaaaac taaaaggttt
     5041 tttctttttg aaaagatcgt tttctttatt attctctagt tttgacggcg gccgcagtga
     5101 ttagacttag tccgttcaaa tcaagcacaa ctctgttcat tgtttcaaca agaattaatt
     5161 caaaaacagg ttcggtgcat aatttgcaaa aaaatattgc agcttctgtg gctcgaacac
     5221 agtacctcca gatttcaggt ttgaaatact tcagtctgac gctctcccag atgagctaaa
     5281 gctgcaataa gaaaacccac gccgggattc gaacccggaa tcctttgatt agaagtcaaa
     5341 agcgataacc atttcgccac gcaggcctac ttgatgggtt tgtaaatggt ctactttttc
     5401 agacctaaca gaaattttaa tgaaagtcat attcttatac aataaaactg tgtcataaaa
     5461 gcagatattc gactttcgta gattatatag gacccaagaa ctaaaattta atgccatatt
     5521 atgcattttt aatctgtaaa agtgttgttt ccaacctatc acaagtacgt tcttgtaact
     5581 tgtgtttgta gggttgcaaa tgaatcataa caacatctca acagaacatg tatagcaaag
     5641 cttagtataa aatcagtgtt ttgagaggca atccaagaat gtttacatca aagtttcaat
     5701 aaatatcgac cgaaactgaa aatcttttta ggttattgtt cacttttttg taaatattta
     5761 aacatttttt ggacctaaaa aaatacaaac accaattacg taccaagaag catctaatca
     5821 actcccagat caccactata catttaaaag tcattggtca ataactatac tcgagtattg
     5881 cctcatcaaa gaaacaatca aatattatag atactcactc catcacgtga taatttcact
     5941 ggtatggaaa agtggaaaat tttataaaaa aaaatttgat gcctttggca tagctgaaac
     6001 ttcggcccaa taggattgga gaatatgttt tcgcagcgtt cttacaatta aattgtggtg
     6061 gaagttcgag acttgcgtaa actattttta atttggagac ggaattccgt ctcgttttag
     6121 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     6181 agtcggtgct ttttttctcg agttttttta tcgagtgttt aaggataatg ataactgaag
     6241 agaagaatta gttttttcaa gtgtataata tagtttctct ctattacctt ttccaataat
     6301 agcattttaa gttttctatt ttattttgta taaaaaacat aatgaaaaat acgtataagt
     6361 aatataaatg agtgtgggat taagtgaata cgagatgttg tagtgataat aggggaaact
     6421 ctttggcgaa actacaagag agagtgatgt gctaataatg aacgaagaaa tatgtgattt
     6481 ttgtatgaaa tttgcaatta ttctgattga atttgggtac ttgacattga atccagaacg
     6541 actatacaaa tgtgctactt tgtcaaaata tcctttttga gaatcggcat atttatggcc
     6601 ctgaatatcg actaccacat tccttttaca acactacgta accttttgag aaagtacaag
     6661 tgaaagaagt atagaattca gtgtttagtt taacgtaagt attactgtgg aacgctttct
     6721 tcgcgacaca agcaacttgt acctgcaccc ttcacacaat ttatttccta aaactactcc
     6781 agtgcgaaaa caatagtgct aaatatgatg atgagagaat tcttaacgaa cggagtagga
     6841 atgtacatac tatcactagt ttccaaataa caaaaataaa aaaaaaaata acatggaact
     6901 tgtattgcta aataaattac tagattttat aagcaataaa aagaatttga aaaggatgct
     6961 tcatcacaac taatagttta gtttctttac ttctcccctg tttactgggt tattttattt
     7021 agattatgct aatataattt tttaatacaa gaatttttat ttttttaatt tatgttgctg
     7081 attgccccta aaatttcaaa ttcctgaaat tccctgagtg acttgaaccc agacacacat
     7141 tcactcactc acacaaacaa atacacaaaa ttagagaacc tgaatttcag attctcaaat
     7201 tccaaaacag caaagccgcg gtggagctcc aattcgccct atagtgagtc gtattacgcg
     7261 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     7321 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     7381 gatcgccctt cccaacagtt gcgcagcctg aatggcga
//
