LOCUS       sequence-431521-        3337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) epegRNA expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3337)
  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 3337)
  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: AA364
FEATURES             Location/Qualifiers
     source          1..3337
                     /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)"
     primer_bind     complement(1247..1263)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1247..1263)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1260..1282)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1271..1287
                     /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(1295..1301,1302..1319,1320..1325))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1340..1361
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1478..1495)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1649..2237)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1729..1748)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2415..3224)
                     /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     3138..3157
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3216..3235
                     /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 tctcacgcgg ttctatctag
      901 ttacgcgtta aaccaactag aatttttttg cctacctgga gcctgagagt tgttcaataa
      961 aataaaaatg tttcgttttt ttgctttcgc cagtatttat tatttttcat caatatgtat
     1021 tcaatttggt atgtatttag taattgtaat atatagacaa tggttttccg ttgacgtaca
     1081 tacatctgac gtgtgtttat ttagacataa tagttatgtt ttcacatctt tttaatgttc
     1141 gcttaatgcg tatgcattct agacaattgt gctcggcaca cgcgtcttca tcggatcccg
     1201 ggcccgtcga ctgcagaggc ctgcatgcaa gcttggtgta atcatggtca tagctgtttc
     1261 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     1321 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     1381 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1441 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1501 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1561 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1621 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1681 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1741 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1801 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1861 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     1921 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     1981 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2041 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2101 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2161 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2221 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2281 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2341 tccttttaaa ttaaaaatga agttttaaat caagcccaat ctgaataatg ttacaaccaa
     2401 ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     2461 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     2521 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     2581 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     2641 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     2701 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     2761 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     2821 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     2881 cctgaatcag gatattcttc taatacctgg aatgctgttt ttccggggat cgcagtggtg
     2941 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     3001 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     3061 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaagcgata gattgtcgca
     3121 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     3181 gaatttaatc gcggcctcga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     3241 ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg
     3301 taacatcaga gattttgaga cacgggccag agctgca
//
