LOCUS       sequence-431480-        3086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) guide expression; positive 
            control cell surface with 2xPP7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3086)
  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 3086)
  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: AA232
FEATURES             Location/Qualifiers
     source          1..3086
                     /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"
     primer_bind     complement(996..1012)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(996..1012)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1009..1031)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1020..1036
                     /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(1044..1050,1051..1068,1069..1074))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1089..1110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1227..1244)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1398..1986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1478..1497)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2164..2973)
                     /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     2887..2906
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2965..2984
                     /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 agagagggcc 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 ccggagcagg catccggcct ccggtttaag
      721 agctatgctg cgaatacgag ggagcagacg atatggcgtc gctcctctcc acgagagcat
      781 atgggctccg tggtctcgta caccatcagg gtacgtatca gacaccatca gggtctgctc
      841 gtattcgcag catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca
      901 ccgagtcggt gcttttttga attcgaacac gcgtcttcat cggatcccgg gcccgtcgac
      961 tgcagaggcc tgcatgcaag cttggtgtaa tcatggtcat agctgtttcc tgtgtgaaat
     1021 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     1081 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     1141 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1201 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1261 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1321 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1381 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1441 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1501 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1561 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1621 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     1681 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     1741 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     1801 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     1861 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     1921 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1981 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2041 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2101 taaaaatgaa gttttaaatc aagcccaatc tgaataatgt tacaaccaat taaccaattc
     2161 tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     2221 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     2281 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     2341 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     2401 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     2461 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     2521 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     2581 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     2641 atattcttct aatacctgga atgctgtttt tccggggatc gcagtggtga gtaaccatgc
     2701 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     2761 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     2821 aaacaactct ggcgcatcgg gcttcccata caagcgatag attgtcgcac ctgattgccc
     2881 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     2941 cggcctcgac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     3001 agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag
     3061 attttgagac acgggccaga gctgca
//
