LOCUS       sequence-381845-        2973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Consensus I-F CRISPR Leader and a CRISPR locus with two consensus 
            I-F repeats, for integration assays..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2973)
  AUTHORS   Santiago-Frangos A, Henriques WS, Wiegand T, Gauvin CC, Buyukyoruk 
            M, Graham AB, Wilkinson RA, Triem L, Neselu K, Eng ET, Lander GC, 
            Wiedenheft B
  TITLE     Structure reveals why genome folding is necessary for site-specific 
            integration of foreign DNA into CRISPR arrays.
  JOURNAL   Nat Struct Mol Biol. 2023 Nov;30(11):1675-1685. doi: 
            10.1038/s41594-023-01097-2. Epub 2023 Sep 14.
  PUBMED    37710013
REFERENCE   2  (bases 1 to 2973)
  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"; doi: 
            "10.1038/s41594-023-01097-2"; journalName: "Nat Struct Mol Biol"; 
            date: "2023-11"; volume: "30"; issue: "11"; pages: "1675-1685"
COMMENT     Sequence Label: pCon-IFl_Con-IFr
FEATURES             Location/Qualifiers
     source          1..2973
                     /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     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    568..587
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    628..647
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(752..768)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(752..768)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(765..787)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    776..792
                     /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(800..806,807..824,825..830))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    845..866
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(983..1000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1154..1742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1234..1253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1913..2704,2705..2773))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2536..2555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2774..2878)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2946..2964
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     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 ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tccatggacc cttttttcgg acgatttaaa aaacccttat aaatcagcaa
      481 gttacgagac ctcgaaaaaa aagggtttct ggcgggaaaa actcaaaaaa cgcttcgaaa
      541 tcaaccggtt ataggttttc ggagctagtt cactgccgta taggcagctt agaaaatcag
      601 ccggacgttg tagtagtcga gcgcggtgtt cactgccgta taggcagctt agaaagccgg
      661 gtggtgactt ctggacggcg ttcgtcaagg atccatcgga tcccgggccc gtcgactgca
      721 gaggcctgca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
      781 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
      841 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
      901 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
      961 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1021 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1081 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1141 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1201 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     1261 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     1321 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     1381 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1441 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     1501 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     1561 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     1621 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     1681 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     1741 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     1801 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     1861 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     1921 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     1981 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     2041 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     2101 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     2161 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     2221 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     2281 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     2341 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     2401 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     2461 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     2521 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     2581 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     2641 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     2701 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     2761 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     2821 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     2881 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     2941 ataaaaatag gcgtatcacg aggccctttc gtc
//
