LOCUS       sequence-431445-        3039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) reverse orientation; guide 
            expression; positive control cell surface.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3039)
  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 3039)
  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: AA083
FEATURES             Location/Qualifiers
     source          1..3039
                     /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(562..810)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(620..639)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(790..810)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(949..965)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(949..965)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(962..984)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    973..989
                     /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(997..1003,1004..1021,1022..1027))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1042..1063
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1180..1197)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1351..1939)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1431..1450)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2117..2926)
                     /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     2840..2859
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2918..2937
                     /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 ttttcaaaaa aaatgggaac
      481 agcagtggta agcccaatct acgacagtag aaattagctc tcagggaaca tgtcaaagta
      541 tctacaagag tagaaattac cggtgtttcg tcctttccac aagatatata aagccaagaa
      601 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
      661 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
      721 tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa
      781 atatgaagga atcatgggaa ataggccctc ttcgaatcta gttgcaaaga tggataaagt
      841 tttaaacaga gaggaatctt tgcagctaat ggaccttcta cacgcgtctt catcggatcc
      901 cgggcccgtc gactgcagag gcctgcatgc aagcttggtg taatcatggt catagctgtt
      961 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     1021 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     1081 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     1141 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     1201 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     1261 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     1321 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     1381 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     1441 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     1501 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     1561 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     1621 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     1681 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     1741 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     1801 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     1861 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     1921 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     1981 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     2041 gatcctttta aattaaaaat gaagttttaa atcaagccca atctgaataa tgttacaacc
     2101 aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2161 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2221 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     2281 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2341 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2401 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2461 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     2521 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2581 cacctgaatc aggatattct tctaatacct ggaatgctgt ttttccgggg atcgcagtgg
     2641 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2701 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2761 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaagcga tagattgtcg
     2821 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2881 tggaatttaa tcgcggcctc gacgtttccc gttgaatatg gctcataaca ccccttgtat
     2941 tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa
     3001 tgtaacatca gagattttga gacacgggcc agagctgca
//
