LOCUS       sequence-431499-        5786 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (Cas protein) NNGG PAM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5786)
  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 5786)
  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: AA301
FEATURES             Location/Qualifiers
     source          1..5786
                     /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"
     primer_bind     complement(3696..3712)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3696..3712)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3709..3731)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3720..3736
                     /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(3744..3750,3751..3768,3769..3774))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3789..3810
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3927..3944)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4098..4686)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4178..4197)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4864..5673)
                     /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     5587..5606
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5665..5684
                     /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 tgaagactgg cagccaggag aaccagcaga agcagaacta catcctgggc
      481 ctggacatcg gaatcaccag cgtcggctac ggactgatcg atagcaagac aagagaagtg
      541 atcgacgccg gcgttagact ctttccagaa gctgatagcg agaacaactc caaccgcaga
      601 agcaagcggg gcgccagacg gttaaaacgg agaagaatcc accggctgaa ccgggtcaaa
      661 gacctgctcg ctgattacca gatgatcgat cttaacaatg ttcctaagag caccgacccc
      721 tacaccatca gagtgaaggg cctccgggag cctctgacaa aagaagaatt cgccatcgcc
      781 ctcctgcata tcgctaagag aagaggcctg cacaacatca gtgtgtccat gggcgacgaa
      841 gagcaggaca atgaactgag caccaagcag cagctgcaaa agaatgccca gcaactgcag
      901 gacaagtatg tgtgcgaact gcagttagaa cggctgacca acatcaacaa ggtcagaggc
      961 gagaagaaca gatttaagac agaggacttt gtgaaagaag tgaaacagct gtgcgaaacc
     1021 cagagacagt accacaacat cgacgaccaa ttcatccagc agtacatcga cctggtgtct
     1081 acaagacggg agtacttcga gggccccggc aacggctctc catacggctg ggacggcgac
     1141 ctgctgaagt ggtacgagaa gctgatgggc agatgcacct atttccccga agaactgagg
     1201 tccgtgaagt acgcctacag cgccgacctc ttcaacgccc tgaacgacct gaacaacctc
     1261 gttgtgacca gggatgacaa tccaaagctt gagtactacg agaagtacca cattattgag
     1321 aacgtgttca agcaaaagaa gaatcccaca ctcaaacaaa tcgccaaaga gatcggcgtg
     1381 caagattacg acatccgggg ctatagaatc acaaagagcg gcaaacctca gttcacctct
     1441 tttaagctgt atcacgacct gaagaacatc ttcgagcagg ccaaatacct ggaagatgtg
     1501 gaaatgctgg acgagatcgc caagatcctg accatctacc aggatgagat tagcatcaag
     1561 aaagccctgg accagctgcc cgaactgctg acagagagcg agaaatctca gatcgcacag
     1621 ctcaccggct atacaggcac ccacagactg agcctgaagt gcatccacat tgtgatcgac
     1681 gagctgtggg agagccccga gaaccagatg gaaatcttta ccagactgaa tctgaaacct
     1741 aagaaggtgg aaatgagcga gatcgacagc atacccacca ccctggtcga cgagttcatc
     1801 ctctcacctg tggtgaagcg ggccttcatc cagagcatca aggtaatcaa cgcagtgatc
     1861 aatcggttcg gcctgccaga ggacatcatc atcgagctgg ccagagaaaa gaatagcaag
     1921 gatcggagaa agttcattaa caagctgcag aaacaaaatg aggccacaag aaagaaaatc
     1981 gaacagctgc tggccaagta cggcaacacc aatgccaagt acatgatcga gaagatcaag
     2041 ctgcacgaca tgcaggaggg caagtgcctg tacagcctgg aggctattcc tctggaggac
     2101 ctgctgagca acccgacaca ctacgaagtt gaccacatta tccccagatc tgtgagcttt
     2161 gacaacagcc tgaacaacaa agtgctggtg aaacaaagcg aaaacagcaa gaagggcaat
     2221 cgcacccctt accagtacct gagcagcaac gagtctaaga ttagctacaa ccagtttaag
     2281 cagcacatcc tgaacctgag caaggccaag gacagaatca gcaagaaaaa aagagatatg
     2341 ctgctggaag agagagatat caacaagttc gaagtgcaga aggaattcat taaccggaac
     2401 ctggtggata cacggtacgc caccagagaa ctgtctaacc tgctgaaaac ctacttcagc
     2461 acccatgact acgccgtgaa ggtgaaaacc atcaacggcg gcttcactaa ccacctgagg
     2521 aaggtgtggg atttcaagaa gcacagaaac cacggctaca agcaccacgc cgaagatgcc
     2581 ctggtgatcg ccaacgccga cttcctgttt aagacacata aggccctgcg gagaaccgat
     2641 aagatcctgg aacaacctgg cctggaagtg aatgatacaa ccgtgaaagt ggacaccgag
     2701 gaaaaatacc aggagctgtt cgagacacct aagcaagtga agaacatcaa gcagttccgg
     2761 gacttcaagt acagccaccg agtggacaag aagcctaacc ggcagcttat caacgacaca
     2821 ctgtactcca ccagagagat tgatggcgaa acctacgtgg tgcagaccct taaggatctg
     2881 tacgccaagg acaacgagaa agtgaagaag ctgttcaccg aaagacctca gaagatcctg
     2941 atgtaccagc acgaccctaa gaccttcgag aaactgatga caatcctgaa ccagtacgct
     3001 gaggccaaga accctctggc tgcttattac gaggacaaag gcgagtacgt gaccaagtac
     3061 gccaagaaag gcaatggacc tgccatccac aagatcaagt atatcgataa gaagcttgga
     3121 tcttacctgg atgttagcaa caagtatcct gagacacaga acaagcttgt gaagctgtcc
     3181 ctgaagagct ttagattcga catctacaag tgtgaacagg gctacaagat ggtgtccatc
     3241 ggatacctgg acgtgctgaa gaaagataac tactactaca tccctaagga caagtacgag
     3301 gccgagaagc agaaaaagaa gatcaaggaa tctgatcttt ttgtgggcag cttctactac
     3361 aacgacctca tcatgtacga ggatgaactg ttcagagtga taggagtgaa cagcgacatc
     3421 aacaatctgg ttgagctaaa catggtcgac attacctaca aggacttctg cgaggtgaac
     3481 aacgtgacag gcgagaaaag aatcaaaaag actatcggca agcgcgtggt cctgatcgag
     3541 aagtacacca cagatattct aggcaacctg tacaagactc ccctgcctaa gaagccccag
     3601 cttatcttca agcggggaga actggggtcc aagtcttcat cggatcccgg gcccgtcgac
     3661 tgcagaggcc tgcatgcaag cttggtgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3721 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3781 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3841 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3901 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3961 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4021 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4081 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4141 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4201 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4261 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4321 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4381 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4441 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4501 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4561 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4621 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4681 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4741 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4801 taaaaatgaa gttttaaatc aagcccaatc tgaataatgt tacaaccaat taaccaattc
     4861 tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     4921 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     4981 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     5041 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     5101 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     5161 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     5221 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     5281 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     5341 atattcttct aatacctgga atgctgtttt tccggggatc gcagtggtga gtaaccatgc
     5401 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     5461 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     5521 aaacaactct ggcgcatcgg gcttcccata caagcgatag attgtcgcac ctgattgccc
     5581 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     5641 cggcctcgac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     5701 agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag
     5761 attttgagac acgggccaga gctgca
//
