LOCUS       sequence-381842-        3059 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Consensus II-A CRISPR Leader and a CRISPR locus with two consensus 
            II-A repeats, for integration assays..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3059)
  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 3059)
  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-IIAl_Con-IIAr
FEATURES             Location/Qualifiers
     source          1..3059
                     /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(838..854)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(838..854)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(851..873)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    862..878
                     /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(886..892,893..910,911..916))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    931..952
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1069..1086)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1240..1828)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1320..1339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1999..2790,2791..2859))
                     /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     2622..2641
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2860..2964)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3032..3050
                     /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 tccatggatc tctttaaata atgtggatgt cctgttttta gaacaaaggg
      481 tggtccagaa cagattccaa tatattttgg acgaaaactt ttatttgagt tatgaaaaag
      541 cttaaattgt tactgattag tggttcattc taaactgaaa tctagctatg gataagtgat
      601 gcgagtacgg aactttggaa aaaaataatt ctccgaggtt ttagagtcat attatttaga
      661 atggtaccaa aacatcagcc ggacgttgta gtagtcgagc gcggtgtttt agagtcatat
      721 tatttagaat ggtaccaaaa cgccgggtgg tgacttctgg acggcgttcg tcaaggatcc
      781 atcggatccc gggcccgtcg actgcagagg cctgcatgca agcttggcgt aatcatggtc
      841 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
      901 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
      961 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     1021 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     1081 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     1141 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     1201 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     1261 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     1321 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     1381 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     1441 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     1501 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     1561 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     1621 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     1681 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     1741 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1801 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     1861 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1921 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1981 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     2041 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     2101 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     2161 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     2221 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     2281 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     2341 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     2401 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     2461 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     2521 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     2581 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     2641 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     2701 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     2761 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     2821 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     2881 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2941 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     3001 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
