LOCUS       sequence-431475-        3008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) Tet-regulatable guide expression;
            positive control cell surface.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3008)
  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 3008)
  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: AA074
FEATURES             Location/Qualifiers
     source          1..3008
                     /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        570..664
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     581..600
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    618..636
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    646..664
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(918..934)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(918..934)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(931..953)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    942..958
                     /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(966..972,973..990,991..996))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1011..1032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1149..1166)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1320..1908)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1400..1419)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2086..2895)
                     /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     2809..2828
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2887..2906
                     /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 agaccgaacg ctgacgtcat caacccgctc caaggaatcg
      481 cgggcccagt gtcactaggc gggaacaccc agcgcgcgtg cgccctggca ggaagatggc
      541 tgtgagggac aggggagtgg cgccctgcaa tatttgcatg tcgctatgtg ttctgggaaa
      601 tcaccataaa cgtgaaatcc ctatcagtga tagagactta taagttccct atcagtgata
      661 gagacaccgg taatttctac tcttgtagat actttgacat gttccctgag agctaatttc
      721 tactgtcgta gattgggctt accactgctg ttcccatttt ttgaattcgc tagcaagctt
      781 ggcgtaacta gatcttgaga caaatggcag tattcatcca caaaggtctt gaaaggagtg
      841 ggaattggac acgcgtcttc atcggatccc gggcccgtcg actgcagagg cctgcatgca
      901 agcttggtgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
      961 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1021 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1081 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1141 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1201 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1261 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     1321 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     1381 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1441 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1501 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     1561 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1621 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1681 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1741 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     1801 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1861 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1921 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1981 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2041 tcaagcccaa tctgaataat gttacaacca attaaccaat tctgattaga aaaactcatc
     2101 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     2161 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     2221 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     2281 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     2341 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     2401 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     2461 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     2521 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     2581 gaatgctgtt tttccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     2641 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     2701 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     2761 gggcttccca tacaagcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     2821 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg acgtttcccg
     2881 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     2941 tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag acacgggcca
     3001 gagctgca
//
