LOCUS       sequence-381846-        2976 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutant 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 2976)
  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 2976)
  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: pX2-IFl_Con-IFr
FEATURES             Location/Qualifiers
     source          1..2976
                     /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"
     primer_bind     complement(755..771)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(755..771)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(768..790)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    779..795
                     /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(803..809,810..827,828..833))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    848..869
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(986..1003)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1157..1745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1237..1256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1916..2707,2708..2776))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPVERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2539..2558
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2777..2881)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2949..2967
                     /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 ctgatcgaaa aaaaagggtt tctggcggga aaaactcaaa aaacgcttcg
      541 aaatcaaccg gttataggtt ttcggagcta gttcactgcc gtgtaggcag ctaagaaaat
      601 cagccggacg ttgtagtagt cgagcgcggt gttcactgcc gtgtaggcag ctaagaaagc
      661 cgggtggtga cttctggacg gcgttcgtca aggatccatc ggatcccggg cccgtcgact
      721 gcagaggcct gcatgcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
      781 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
      841 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
      901 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
      961 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1021 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1081 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1141 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1201 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1261 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1321 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1381 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1441 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1501 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1561 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     1621 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1681 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1741 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1801 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1861 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     1921 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     1981 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2041 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2101 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2161 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2221 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2281 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2341 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2401 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2461 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2521 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     2581 ttggaaaacg ttctacgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2641 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2701 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2761 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2821 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2881 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     2941 cctataaaaa taggcgtatc acgaggccct ttcgtc
//
