LOCUS       sequence-431522-        3375 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 3375)
  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 3375)
  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: AA363
FEATURES             Location/Qualifiers
     source          1..3375
                     /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..864
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        886..961
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1285..1301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1285..1301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1298..1320)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1309..1325
                     /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(1333..1339,1340..1357,1358..1363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1378..1399
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1516..1533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1687..2275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1767..1786)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2453..3262)
                     /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     3176..3195
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3254..3273
                     /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 agattttttg ctcacctgtg attgctccta ctcaaataca
      481 aaaacatcaa attttctgtc aataaagcat atttatttat atttatttta caggaaagaa
      541 ttccttttaa agtgtatttt aacctataat gaaaaacgat taaaaaaaat acataaaata
      601 attcgaaaat ttttgaatag cccaggttga taaaaattca tttcatacgt tttataactt
      661 atgcccctaa gtattttttg accatagtgt ttcaattcta cattaatttt acagagtaga
      721 atgaaacgcc acctactcag ccaagaggcg aaaaggttag ctcgccaagc agagagggcg
      781 ccagtgctca ctacttttta taattctcaa cttctttttc cagactcagt tcgtatatat
      841 agacctattt tcaatttaac gtcgggagac gcttttttcg tctcagtttt agagctagaa
      901 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      961 cttttttgcc tacctggagc ctgagagttg ttcaataaaa taaaaatgtt tcgttttttt
     1021 gctttcgcca gtatttatta tttttcatca atatgtattc aatttggtat gtatttagta
     1081 attgtaatat atagacaatg gttttccgtt gacgtacata catctgacgt gtgtttattt
     1141 agacataata gttatgtttt cacatctttt taatgttcgc ttaatgcgta tgcattctag
     1201 acaattgtgc tcggcacacg cgtcttcatc ggatcccggg cccgtcgact gcagaggcct
     1261 gcatgcaagc ttggtgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     1321 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     1381 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     1441 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1501 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     1561 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     1621 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     1681 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     1741 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     1801 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     1861 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     1921 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     1981 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2041 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2101 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2161 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2221 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2281 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2341 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     2401 ttttaaatca agcccaatct gaataatgtt acaaccaatt aaccaattct gattagaaaa
     2461 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     2521 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     2581 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     2641 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     2701 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     2761 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2821 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2881 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2941 atacctggaa tgctgttttt ccggggatcg cagtggtgag taaccatgca tcatcaggag
     3001 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     3061 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     3121 gcgcatcggg cttcccatac aagcgataga ttgtcgcacc tgattgcccg acattatcgc
     3181 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctcgacg
     3241 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     3301 ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca
     3361 cgggccagag ctgca
//
