LOCUS       sequence-367528-        3460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shu sgRNA plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3460)
  AUTHORS   Meng Q, Stoyko D, Andrews CM, Konstantinidou P, Genzor P, O T, 
            Elchert AR, Benner L, Sobti S, Katz EY, Haase AD
  TITLE     Functional editing of endogenous genes through rapid selection of 
            cell pools (Rapid generation of endogenously tagged genes in 
            Drosophila ovarian somatic sheath cells).
  JOURNAL   Nucleic Acids Res. 2022 May 27. pii: 6594084. doi: 
            10.1093/nar/gkac448.
  PUBMED    35639929
REFERENCE   2  (bases 1 to 3460)
  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. 2022 May 27. pii: 6594084. doi: 10.1093/nar/gkac448."
COMMENT     Sequence Label: Shu_sgRNA
FEATURES             Location/Qualifiers
     source          1..3460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..138
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     130..147
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     131..147
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     154..173
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        154..172
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     197..213
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     198..214
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        226..625
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        646..721
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(750..766)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(750..766)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(803..821)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(842..858)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(842..858)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(855..877)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    866..882
                     /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)."
     promoter        complement(join(890..896,897..914,915..920))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    935..956
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1073..1090)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1244..1832)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1324..1343)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2003..2794,2795..2863))
                     /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     2626..2645
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2864..2968)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2994..3449)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3131..3152)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3343..3362
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
       61 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
      121 tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg
      181 ggtaccgggc cccccctcga ggtcgacggt atcgataagc ttgatgttcg acttgcagcc
      241 tgaaatacgg cacgagtagg aaaagccgag tcaaatgccg aatgcagagt ctcattacag
      301 cacaatcaac tcaagaaaaa ctcgacactt ttttaccatt tgcacttaaa tcctttttta
      361 ttcgttatgt atactttttt tggtccctaa ccaaaacaaa accaaactct cttagtcgtg
      421 cctctatatt taaaactatc aatttattat agtcaataaa tcgaactgtg ttttcaacaa
      481 acgaacaata ggacactttg attctaaagg aaattttgaa aatcttaagc agagggttct
      541 taagaccatt tgccaattct tataattctc aactgctctt tcctgatgtt gatcatttat
      601 ataggtatgt tttcctcaat acttcgatag ctttagcgaa tcgatgtttt agagctagaa
      661 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      721 cttttttatc gaattcctgc agcccggggg atccactagt tctagagcgg ccgccaccgc
      781 ggtggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat
      841 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
      901 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
      961 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1021 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     1081 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     1141 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     1201 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1261 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1321 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1381 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     1441 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     1501 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     1561 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     1621 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     1681 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     1741 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     1801 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     1861 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     1921 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     1981 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2041 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     2101 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     2161 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     2221 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     2281 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     2341 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     2401 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     2461 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     2521 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     2581 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     2641 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     2701 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     2761 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     2821 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     2881 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     2941 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     3001 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3061 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     3121 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3181 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3241 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3301 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3361 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3421 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
