LOCUS       sequence-431534-        5765 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 5765)
  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 5765)
  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: AA299
FEATURES             Location/Qualifiers
     source          1..5765
                     /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(3675..3691)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3675..3691)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3688..3710)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3699..3715
                     /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(3723..3729,3730..3747,3748..3753))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3768..3789
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3906..3923)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4077..4665)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4157..4176)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4843..5652)
                     /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     5566..5585
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5644..5663
                     /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 cagcaaccaa aaattcatac tgggactgga catcggaatc
      481 accagcgtgg gctacggcct gatcgactac gagacaaaga atatcatcga tgccggcgtt
      541 agactgttcc ccgaggccaa cgtggaaaac aacgagggaa gaaggtccaa acgtggaagc
      601 aggagactga agcgacgccg cattcacaga cttgaacggg tgaagaagct gctcgaggat
      661 tataatctgc tggatcagtc ccagattcct cagtctacaa acccctacgc catccgcgtg
      721 aagggcctgt ctgaagccct gagcaaggac gaactcgtga ttgccctgct ccatatcgcc
      781 aagagaagag gcatccacaa gatcgacgtg atcgacagca acgacgacgt ggggaacgag
      841 ctcagcacca aggaacagct gaataagaac agcaagctgc tgaaagacaa atttgtgtgc
      901 cagatccagc tggaaagaat gaatgagggc caggtgcggg gagagaaaaa ccggttcaag
      961 accgctgata tcatcaagga aatcatccag ctgctgaatg tgcagaagaa cttccaccag
     1021 ctggacgaga acttcatcaa caagtacatc gaactggttg agatgagaag agaatacttc
     1081 gagggccccg gcaagggcag tccatatggc tgggaaggcg accctaaggc ttggtacgag
     1141 acactgatgg gccactgcac ctacttccca gatgagctga gaagcgtgaa atacgcctac
     1201 agtgccgacc tgttcaacgc tctgaacgac ctgaacaacc tggtcatcca aagagatgga
     1261 ctgtctaagc tcgagtatca tgagaagtat cacatcatcg agaacgtgtt caagcagaag
     1321 aagaaaccta cactgaagca gatcgccaat gagatcaatg tcaaccctga agatatcaag
     1381 ggctacagaa tcacaaagtc tggcaagccc cagtttaccg agtttaagct ctaccacgac
     1441 ctgaaaagcg tgctgtttga ccagagcatc ctggagaacg aggacgtgct ggaccagatc
     1501 gctgagatcc tgaccatcta ccaggacaag gatagcatca aatctaagct gacggaactg
     1561 gacatcctgc tgaacgagga agataaggaa aacatcgccc agctgactgg ctacaccggg
     1621 acccaccggc tcagcctgaa atgcatccgg ctggtcctgg aagagcagtg gtattctagc
     1681 cggaatcaga tggaaatctt cacacacctg aacattaagc ctaagaagat caacctgaca
     1741 gccgccaaca agatcccgaa ggctatgatc gacgagttca tcctgagccc tgtggtgaag
     1801 aggaccttcg gccaggccat taaccttatt aacaagatca tagaaaagta cggcgtgcct
     1861 gaagatatca tcatcgagct ggccagagaa aataatagca aggacaagca gaagttcatc
     1921 aatgagatgc agaaaaagaa cgagaacacc agaaagagaa ttaacgaaat catcggcaag
     1981 tatggcaacc agaacgccaa gagactggtc gagaagatta gactgcacga cgagcaggag
     2041 ggcaagtgcc tgtactcact ggaaagcatc cctctggagg acctgctgaa caaccccaac
     2101 cactacgagg tggaccacat cattccaaga tctgtgtcct tcgacaactc ttaccacaac
     2161 aaagtgctcg tgaagcagag cgagaactcc aaaaaatcca acctgacccc ttaccagtac
     2221 tttaacagcg gcaagtccaa gctctcttac aaccagttta aacaacacat cctgaacctg
     2281 agcaagtccc aggatagaat cagcaaaaaa aagaaagagt atctgctgga agaacgggac
     2341 atcaacaagt tcgaggtgca aaaagagttc atcaatagaa acctggtgga tacccggtac
     2401 gccacaagag agctgacaaa ctacctgaag gcctacttca gcgccaacaa tatgaacgtg
     2461 aaggtgaaaa cgatcaacgg cagcttcacc gattacctgc ggaaagtgtg gaagtttaag
     2521 aaggaacgga accacggcta caagcaccac gccgaggacg ccctgattat cgctaatgcc
     2581 gatttcctgt tcaaagagaa caagaagctg aaagccgtga actctgtgct ggaaaaacct
     2641 gagatcgaga gcaagcagct ggatatccag gtggatagcg aggataacta cagcgaaatg
     2701 ttcatcatcc ctaagcaggt ccaggacatc aaggacttca gaaacttcaa gtacagccac
     2761 agagtggaca agaagcctaa cagacagctg atcaacgata cactgtacag cacccggaag
     2821 aaggacaact ccacctacat cgtgcagacc atcaaagata tctatgccaa agataatacc
     2881 accctgaaga agcagtttga caagtcaccc gagaagttcc tcatgtacca acacgatccg
     2941 cggaccttcg agaagttgga agtgatcatg aagcagtacg ctaatgagaa gaatcctctg
     3001 gccaagtacc acgaggaaac aggcgagtac ctgaccaaat acagcaaaaa aaacaacggc
     3061 cctatcgtga aaagcctgaa gtacattgga aacaagctgg gcagccacct agatgtgacc
     3121 caccagttca agagcagcac caagaagttg gtgaagctga gcatcaagcc ttatagattc
     3181 gacgtctacc tgaccgacaa gggatataag ttcatcacca tcagctacct ggacgtgctg
     3241 aagaaagaca attactacta catacccgaa cagaagtacg acaagctcaa actgggcaag
     3301 gccatcgaca aaaacgccaa gtttatcgct agcttctaca agaatgatct gatcaagctg
     3361 gacggcgaga tctacaagat catcggcgtg aatagcgaca ccagaaacat gatcgaactg
     3421 gatctgcctg acatcagata caaagaatac tgcgagctga acaatatcaa gggcgaacct
     3481 agaatcaaaa agaccatcgg caaaaaggtg aatagcatcg aaaaactgac aaccgacgtg
     3541 ctgggcaacg tgttcaccaa cacccagtac acaaaacctc agctgctgtt caagcgagga
     3601 aatgggtcca agtcttcatc ggatcccggg cccgtcgact gcagaggcct gcatgcaagc
     3661 ttggtgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3721 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3781 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3841 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3901 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3961 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4021 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4081 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4141 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4201 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4261 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4321 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4381 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4441 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4501 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4561 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4621 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4681 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4741 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4801 agcccaatct gaataatgtt acaaccaatt aaccaattct gattagaaaa actcatcgag
     4861 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     4921 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     4981 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     5041 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     5101 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     5161 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     5221 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     5281 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     5341 tgctgttttt ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     5401 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     5461 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     5521 cttcccatac aagcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     5581 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctcgacg tttcccgttg
     5641 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     5701 tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca cgggccagag
     5761 ctgca
//
