LOCUS       sequence-431474-        3001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) guide expression; reverse 
            orientation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3001)
  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 3001)
  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: AA054
FEATURES             Location/Qualifiers
     source          1..3001
                     /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        complement(524..772)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(582..601)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(752..772)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(911..927)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(911..927)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(924..946)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    935..951
                     /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(959..965,966..983,984..989))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1004..1025
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1142..1159)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1313..1901)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1393..1412)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2079..2888)
                     /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     2802..2821
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2880..2899
                     /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 tgaagacgtc agaaccgact cggtgccact ttttcaagac cagattcaga
      481 gacgagaaaa aacgcgtctc catctacaag agtagaaatt accggtgttt cgtcctttcc
      541 acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc atatgatagt
      601 ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac tttctacgtc
      661 acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt atctctctaa
      721 cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc tcttcgaatc
      781 tagttgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc
      841 tacacgcgtc ttcatcggat cccgggcccg tcgactgcag aggcctgcat gcaagcttgg
      901 tgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
      961 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1021 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     1081 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     1141 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1201 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1261 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1321 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1381 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1441 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1501 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1561 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1621 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1681 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1741 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1801 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1861 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     1921 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     1981 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaagcc
     2041 caatctgaat aatgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc
     2101 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     2161 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     2221 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     2281 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     2341 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     2401 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     2461 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     2521 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     2581 gtttttccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     2641 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     2701 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     2761 ccatacaagc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     2821 ccatataaat cagcatccat gttggaattt aatcgcggcc tcgacgtttc ccgttgaata
     2881 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat
     2941 gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacggg ccagagctgc
     3001 a
//
