LOCUS       sequence-206074-        2567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing an RFP dropout used to construct randomized PAM 
            library.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2567)
  AUTHORS   Maxwell CS, Jacobsen T, Marshall R, Noireaux V, Beisel CL
  TITLE     A detailed cell-free transcription-translation-based assay to 
            decipher CRISPR protospacer-adjacent motifs.
  JOURNAL   Methods. 2018 Jul 1;143:48-57. doi: 10.1016/j.ymeth.2018.02.016. 
            Epub 2018 Feb 24.
  PUBMED    29486239
REFERENCE   2  (bases 1 to 2567)
  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: "Methods."; 
            date: "2018-07-1"; volume: "143:48-57. doi"; pages: " 
            10.1016/j.ymeth.2018.02.016. Epub 2018 Feb 24"
COMMENT     Sequence Label: CSM160
FEATURES             Location/Qualifiers
     source          1..2567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     528..547
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      795..1383
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1284..1303
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1500..1518
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1525..1543
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1561..1572
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1579..2256
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2274..2345
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2361..2388
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2416..2450)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(join(2521..2567,1..613))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca
       61 tcagcacctt gtcgccttgc gtataatatt tgcccatggt gaaaacgggg gcgaagaagt
      121 tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaaa
      181 cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg
      241 ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga
      301 gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc
      361 atatcaccag ctcaccgtct ttcattgcca tacgaaattc cggatgagca ttcatcaggc
      421 gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta
      481 aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa
      541 atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga
      601 tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc
      661 ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc ccgatcaatc
      721 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
      781 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
      841 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      901 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
      961 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1021 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     1081 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1141 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     1201 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     1261 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     1321 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     1381 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     1441 atgttctttc ctgcgttatc ccctgattct gtggataacc gtagtcggaa cgtgagacct
     1501 ccctatcagt gatagagatt gacatcccta tcagtgatag agatactgag cacggatctg
     1561 aaagaggaga aaggatctat ggcgagtagc gaagacgtta tcaaagagtt catgcgtttc
     1621 aaagttcgta tggaaggttc cgttaacggt cacgagttcg aaatcgaagg tgaaggtgaa
     1681 ggtcgtccgt acgaaggtac tcagaccgct aaactgaaag ttaccaaagg tggtccgctg
     1741 ccgttcgctt gggacatcct gtccccgcag ttccagtacg gttccaaagc ttacgttaaa
     1801 cacccggctg acatcccgga ctacctgaaa ctgtccttcc cggaaggttt caaatgggaa
     1861 cgtgttatga acttcgaaga cggtggtgtt gttaccgtta cccaggactc ctccctgcaa
     1921 gacggtgagt tcatctacaa agttaaactg cgtggtacta acttcccgtc cgacggtccg
     1981 gttatgcaga aaaaaaccat gggttgggaa gcttccaccg aacgtatgta cccggaagac
     2041 ggtgctctga aaggtgaaat caaaatgcgt ctgaaactga aagacggtgg tcactacgac
     2101 gctgaagtta aaaccaccta catggctaaa aaaccggttc agctgccggg tgcttacaaa
     2161 accgacatca aactggacat cacctcccac aacgaagact acaccatcgt tgaacagtac
     2221 gaacgtgctg aaggtcgtca ctccaccggt gcttaataag gatctccagg catcaaataa
     2281 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     2341 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataggtctc
     2401 agctggacca gaccaataaa aaacgcccgg cggcaaccga gcgttctgaa caaatccaga
     2461 tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct cgatatcaaa
     2521 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaa
//
