LOCUS       sequence-431512-        2972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) guide expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2972)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 2972)
  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: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA304
FEATURES             Location/Qualifiers
     source          1..2972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        444..692
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     444..464
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     615..634
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        714..789
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     primer_bind     complement(882..898)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(882..898)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(895..917)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    906..922
                     /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(930..936,937..954,955..960))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    975..996
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1113..1130)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1284..1872)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1364..1383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2050..2859)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2773..2792
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2851..2870
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagacgga agcgagggcc tatttcccat gattccttca tatttgcata
      481 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
      541 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      601 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      661 ctttatatat cttgtggaaa ggacgaaaca ccggagacgc ttttttcgtc tcagttttag
      721 tactctggaa acagaatcta ctaaaacaag gcaaaatgcc gtgtttatct cgtcaacttg
      781 ttggcgagat ttttttgttt aaaccgtcca gtcttcggaa ccaaatcgga tcccgggccc
      841 gtcgactgca gaggcctgca tgcaagcttg gtgtaatcat ggtcatagct gtttcctgtg
      901 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
      961 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     1021 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     1081 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1141 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1201 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1261 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1321 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1381 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1441 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1501 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1561 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1621 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     1681 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     1741 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     1801 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     1861 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     1921 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     1981 ttaaattaaa aatgaagttt taaatcaagc ccaatctgaa taatgttaca accaattaac
     2041 caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     2101 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     2161 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     2221 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     2281 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     2341 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     2401 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     2461 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     2521 atcaggatat tcttctaata cctggaatgc tgtttttccg gggatcgcag tggtgagtaa
     2581 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     2641 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     2701 tttcagaaac aactctggcg catcgggctt cccatacaag cgatagattg tcgcacctga
     2761 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     2821 taatcgcggc ctcgacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     2881 tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca
     2941 tcagagattt tgagacacgg gccagagctg ca
//
