LOCUS       sequence-431533-        5849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (Cas protein) NNNNCC PAM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5849)
  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 5849)
  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: AA296
FEATURES             Location/Qualifiers
     source          1..5849
                     /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(3759..3775)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3759..3775)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3772..3794)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3783..3799
                     /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(3807..3813,3814..3831,3832..3837))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3852..3873
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3990..4007)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4161..4749)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4241..4260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4927..5736)
                     /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     5650..5669
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5728..5747
                     /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 cagcgccgcc ttcaagccta acccaatcaa ttacatcctg
      481 ggactggaca tcggaatcgc atccgtggga tgggctatgg tggagatcga cgaggaggag
      541 aatcctatcc ggctgatcga tctgggcgtg agagtgtttg agagggccga ggtgccaaag
      601 accggcgatt ctctggctat ggcccggaga ctggcacgga gcgtgaggcg cctgacacgg
      661 agaagggcac acaggctgct gagggcacgc cggctgctga agagagaggg cgtgctgcag
      721 gcagcagact tcgatgagaa tggcctgatc aagagcctgc caaacacccc ctggcagctg
      781 agagcagccg ccctggacag gaagctgaca ccactggagt ggtctgccgt gctgctgcac
      841 ctgatcaagc accgcggcta cctgagccag cggaagaacg agggagagac agcagacaag
      901 gagctgggcg ccctgctgaa gggagtggcc aacaatgccc acgccctgca gaccggcgat
      961 ttcaggacac ctgccgagct ggccctgaat aagtttgaga aggagtccgg ccacatcaga
     1021 aaccagaggg gcgactatag ccacaccttc tcccgcaagg atctgcaggc cgagctgatc
     1081 ctgctgttcg agaagcagaa ggagtttggc aatccacacg tgagcggagg cctgaaggag
     1141 ggaatcgaga ccctgctgat gacacagagg cctgccctgt ccggcgacgc agtgcagaag
     1201 atgctgggac actgcacctt cgagcctgca gagccaaagg ccgccaagaa cacctacaca
     1261 gccgagcggt ttatctggct gacaaagctg aacaatctga gaatcctgga gcagggatcc
     1321 gagaggccac tgaccgacac agagagggcc accctgatgg atgagcctta ccggaagtct
     1381 aagctgacat atgcccaggc cagaaagctg ctgggcctgg aggacaccgc cttctttaag
     1441 ggcctgagat acggcaagga taatgccgag gcctccacac tgatggagat gaaggcctat
     1501 cacgccatct ctcgcgccct ggagaaggag ggcctgaagg acaagaagtc ccccctgaac
     1561 ctgagctccg agctgcagga tgagatcggc accgccttct ctctgtttaa gaccgacgag
     1621 gatatcacag gccgcctgaa ggacagggtg cagcctgaga tcctggaggc cctgctgaag
     1681 cacatctctt tcgataagtt tgtgcagatc agcctgaagg ccctgagaag gatcgtgcca
     1741 ctgatggagc agggcaagcg gtacgacgag gcctgcgccg agatctacgg cgatcactat
     1801 ggcaagaaga acacagagga gaagatctat ctgcccccta tccctgccga cgagatcaga
     1861 aatcctgtgg tgctgagggc cctgtcccag gcaagaaaag tgatcaacgg agtggtgcgc
     1921 cggtacggat ctccagcccg gatccacatc gagaccgcca gagaagtggg caagagcttc
     1981 aaggaccgga aggagatcga gaagagacag gaggagaatc gcaaggatcg ggagaaggcc
     2041 gccgccaagt ttagggagta cttccctaac tttgtgggcg agccaaagtc taaggacatc
     2101 ctgaagctgc gcctgtacga gcagcagcac ggcaagtgtc tgtatagcgg caaggagatc
     2161 aatctggtgc ggctgaacga gaagggctat gtggagatcg atcacgccct gcctttctcc
     2221 agaacctggg acgattcttt taacaataag gtgctggtgc tgggcagcga gaaccagaat
     2281 aagggcaatc agacaccata cgagtatttc aatggcaagg acaactccag ggagtggcag
     2341 gagttcaagg cccgcgtgga gacctctaga tttcccagga gcaagaagca gcggatcctg
     2401 ctgcagaagt tcgacgagga tggctttaag gagtgcaacc tgaatgacac cagatacgtg
     2461 aaccggttcc tgtgccagtt tgtggccgat cacatcctgc tgaccggcaa gggcaagaga
     2521 agggtgttcg cctctaatgg ccagatcaca aacctgctga ggggattttg gggactgagg
     2581 aaggtgcggg cagagaatga cagacaccac gcactggatg cagtggtggt ggcatgcagc
     2641 accgtggcaa tgcagcagaa gatcacaaga ttcgtgaggt ataaggagat gaacgccttt
     2701 gacggcaaga ccatcgataa ggagacaggc aaggtgctgc accagaaaac ccacttcccc
     2761 cagccttggg agttctttgc ccaggaagtg atgatccggg tgttcggcaa gccagacggc
     2821 aagcctgagt ttgaggaggc cgatacccca gagaagctga ggacactgct ggcagagaag
     2881 ctgtctagca ggccagaggc agtgcacgag tacgtgaccc cactgttcgt gtccagggca
     2941 cccaatcgga agatgtctgg cgcccacaag gacacactga gaagcgccaa gaggtttgtg
     3001 aagcacaacg agaagatctc cgtgaagaga gtgtggctga ccgagatcaa gctggccgat
     3061 ctggagaaca tggtgaatta caagaacggc agggagatcg agctgtatga ggccctgaag
     3121 gcaaggctgg aggcctacgg aggaaatgcc aagcaggcct tcgacccaaa ggataacccc
     3181 ttttataaga agggaggaca gctggtgaag gccgtgcggg tggagaaaac ccaggagagc
     3241 ggcgtgctgc tgaataagaa gaacgcctac acaatcgccg acaatggcga tatggtgaga
     3301 gtggacgtgt tctgtaaggt ggataagaag ggcaagaatc agtactttat cgtgcctatc
     3361 tatgcctggc aggtggccga gaacatcctg ccagacatcg attgcaaggg ctacagaatc
     3421 gacgatagct atacattctg tttttccctg cacaagtatg acctgatcgc cttccagaag
     3481 gatgagaagt ccaaggtgga gtttgcctac tatatcaatt gcgactcctc taacggcagg
     3541 ttctacctgg cctggcacga taagggcagc aaggagcagc agtttcgcat ctccacccag
     3601 aatctggtgc tgatccagaa gtatcaggtg aacgagctgg gcaaggagat caggccatgt
     3661 cggctgaaga agcgcccacc cgtgcggggg tccaagtctt catcggatcc cgggcccgtc
     3721 gactgcagag gcctgcatgc aagcttggtg taatcatggt catagctgtt tcctgtgtga
     3781 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3841 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3901 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3961 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4021 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4081 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4141 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4201 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4261 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4321 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4381 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4441 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4501 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4561 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4621 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4681 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4741 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4801 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4861 aattaaaaat gaagttttaa atcaagccca atctgaataa tgttacaacc aattaaccaa
     4921 ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     4981 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     5041 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     5101 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     5161 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     5221 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     5281 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     5341 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     5401 aggatattct tctaatacct ggaatgctgt ttttccgggg atcgcagtgg tgagtaacca
     5461 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     5521 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     5581 cagaaacaac tctggcgcat cgggcttccc atacaagcga tagattgtcg cacctgattg
     5641 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     5701 tcgcggcctc gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     5761 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca
     5821 gagattttga gacacgggcc agagctgca
//
