LOCUS       sequence-431418-        3128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) for dual expression sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3128)
  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 3128)
  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: AA151
FEATURES             Location/Qualifiers
     source          1..3128
                     /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        458..706
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     458..478
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     629..648
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        complement(748..962)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(808..827)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(1038..1054)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1038..1054)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1051..1073)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1062..1078
                     /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(1086..1092,1093..1110,1111..1116))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1131..1152
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1269..1286)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1440..2028)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1520..1539)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2206..3015)
                     /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     2929..2948
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3007..3026
                     /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 agattaatta aggtaccgag ggcctatttc ccatgattcc
      481 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
      541 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
      601 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
      661 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggag acgcgttttt
      721 tgcgcgcaaa aacgtctccg ggaaagagtg gtctcataca gaacttataa gattcccaaa
      781 tccaaagaca tttcacgttt atggtgattt cccagaacac atagcgacat gcaaatattg
      841 cagggcgcca ctcccctgtc cctcacagcc atcttcctgc cagggcgcac gcgcgctggg
      901 tgttcccgcc tagtgacact gggcccgcga ttccttggag cgggttgatg acgtcagcgt
      961 tcgaattcac acgcgtcttc atcggatccc gggcccgtcg actgcagagg cctgcatgca
     1021 agcttggtgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     1081 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1141 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1201 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1261 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1321 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1381 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     1441 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     1501 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1561 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1621 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     1681 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1741 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1801 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1861 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     1921 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1981 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2041 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2101 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2161 tcaagcccaa tctgaataat gttacaacca attaaccaat tctgattaga aaaactcatc
     2221 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     2281 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     2341 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     2401 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     2461 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     2521 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     2581 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     2641 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     2701 gaatgctgtt tttccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     2761 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     2821 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     2881 gggcttccca tacaagcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     2941 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg acgtttcccg
     3001 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     3061 tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag acacgggcca
     3121 gagctgca
//
