LOCUS       sequence-367529-        3460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GLE 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: GLE_sgRNA
FEATURES             Location/Qualifiers
     source          1..3460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..24
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5..23
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     48..64
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     49..65
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        77..476
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        497..572
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(601..617)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(601..617)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(654..672)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(693..709)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(693..709)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(706..728)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    717..733
                     /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(741..747,748..765,766..771))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    786..807
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(924..941)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1095..1683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1175..1194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1854..2645,2646..2714))
                     /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     2477..2496
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2715..2819)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2845..3300)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2982..3003)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3194..3213
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3427..3449
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3441..3458
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3442..3458
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 attgtaatac gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgacgg
       61 tatcgataag cttgatgttc gacttgcagc ctgaaatacg gcacgagtag gaaaagccga
      121 gtcaaatgcc gaatgcagag tctcattaca gcacaatcaa ctcaagaaaa actcgacact
      181 tttttaccat ttgcacttaa atcctttttt attcgttatg tatacttttt ttggtcccta
      241 accaaaacaa aaccaaactc tcttagtcgt gcctctatat ttaaaactat caatttatta
      301 tagtcaataa atcgaactgt gttttcaaca aacgaacaat aggacacttt gattctaaag
      361 gaaattttga aaatcttaag cagagggttc ttaagaccat ttgccaattc ttataattct
      421 caactgctct ttcctgatgt tgatcattta tataggtatg ttttcctcaa tacttcgcgt
      481 atccccttaa caataagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
      541 atcaacttga aaaagtggca ccgagtcggt gcttttttat cgaattcctg cagcccgggg
      601 gatccactag ttctagagcg gccgccaccg cggtggagct ccagcttttg ttccctttag
      661 tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
      721 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
      781 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
      841 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
      901 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
      961 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     1021 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1081 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     1141 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     1201 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     1261 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     1321 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     1381 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     1441 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1501 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     1561 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     1621 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1681 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1741 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     1801 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     1861 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1921 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1981 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2041 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2101 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     2161 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     2221 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     2281 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     2341 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     2401 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     2461 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     2521 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     2581 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     2641 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2701 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     2761 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     2821 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     2881 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     2941 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3001 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3061 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3121 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3181 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3241 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3301 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     3361 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     3421 ggttttccca gtcacgacgt tgtaaaacga cggccagtga
//
