LOCUS       sequence-372765-        4575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG inducible mCherry with sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4575)
  AUTHORS   Kim SK, Kim H, Woo SG, Kim TH, Rha E, Kwon KK, Lee H, Lee SG, Lee DH
  TITLE     CRISPRi-based programmable logic inverter cascade for 
            antibiotic-free selection and maintenance of multiple plasmids.
  JOURNAL   Nucleic Acids Res. 2022 Dec 9;50(22):13155-13171. doi: 
            10.1093/nar/gkac1104.
  PUBMED    36511859
REFERENCE   2  (bases 1 to 4575)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2022-12-9- 9"; volume: "50"; issue: 
            "22"; pages: "13155-13171"
COMMENT     Sequence Label: pA-RFP-rG2
FEATURES             Location/Qualifiers
     source          1..4575
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(885..1544)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1459..1478
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1545..1647)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2039..2056)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2173..2718)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2253..2272)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(2894..2899,2900..2916,2917..2923)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2931..2947
                     /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)."
     CDS             2996..3706
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3141..3159)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3409..3428
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3755..3826
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3842..3869
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        3919..3953
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        3974..4049
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(4098..4115)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4268..4354
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4446..4473
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tcgggcagcg ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc gttgaggacc
       61 cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga
      121 acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc
      181 ggtttccgtg tttcgtaaag tctggaaacg cggaagtccc ctacgtgctg ctgaagttgc
      241 ccgcaacaga gagtggaacc aaccggtgat accacgatac tatgactgag agtcaacgcc
      301 atgagcggcc tcatttctta ttctgagtta caacagtccg caccgctgcc ggtagctcct
      361 tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc
      421 aactcgtagg acaggtgccg gcagcgccca acagtccccc ggccacgggg cctgccacca
      481 tacccacgcc gaaacaagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct
      541 gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctaaccg tttttatcag
      601 gctctgggag gcagaataaa tgatcatatc gtcaattatt acctccacgg ggagagcctg
      661 agcaaactgg cctcaggcat ttgagaagca cacggtcaca ctgcttccgg tagtcaataa
      721 accggtaaac cagcaataga cataagcggc tatttaacga ccctgccctg aaccgacgac
      781 cgggtcgaat ttgctttcga atttctgcca ttcatccgct tattatcact tattcaggcg
      841 tagcaaccag gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc
      901 cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa
      961 acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat
     1021 ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa
     1081 ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta
     1141 gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac
     1201 tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg
     1261 aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc
     1321 atacggaact ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa
     1381 aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc
     1441 tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat
     1501 tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct
     1561 cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga
     1621 aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt ggcccagggc
     1681 ttcccggtat caacagggac accaggattt atttattctg cgaagtgatc ttccgtcaca
     1741 ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca acttactgat ttagtgtatg
     1801 atggtgtttt tgaggtgctc cagtggcttc tgtttctatc agctgtccct cctgttcagc
     1861 tactgacggg gtggtgcgta acggcaaaag caccgccgga catcagcgct agcggagtgt
     1921 atactggctt actatgttgg cactgatgag ggtgtcagtg aagtgcttca tgtggcagga
     1981 gaaaaaaggc tgcaccggtg cgtcagcaga atatgtgata caggatatat tccgcttcct
     2041 cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg gcttacgaac
     2101 ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg agagggccgc
     2161 ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa tctgacgctc
     2221 aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggcgg
     2281 ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg
     2341 gccgcgtttg tctcattcca cgcctgacac tcagttccgg gtaggcagtt cgctccaagc
     2401 tggactgtat gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc ggtaactatc
     2461 gtcttgagtc caacccggaa agacatgcaa aagcaccact ggcagcagcc actggtaatt
     2521 gatttagagg agttagtctt gaagtcatgc gccggttaag gctaaactga aaggacaagt
     2581 tttggtgact gcgctcctcc aagccagtta cctcggttca aagagttggt agctcagaga
     2641 accttcgaaa aaccgccctg caaggcggtt ttttcgtttt cagagcaaga gattacgcgc
     2701 agaccaaaac gatctcaaga agatcatctt attaatcaga taaaatattt caagatttca
     2761 gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ctaattcgtg tcgctcaagg
     2821 cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt
     2881 ctgaaatgag ctgttgacaa ttaatcatcc ggctcgtata atgtgtggaa ttgtgagcgg
     2941 ataacaattt cgaattcaaa agatctatcg atcgaggatc caggaggtac aatcaatggt
     3001 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     3061 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     3121 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     3181 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     3241 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     3301 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     3361 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     3421 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     3481 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     3541 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     3601 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     3661 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaaggta cctctagagt
     3721 cgacctgcag gcatgcataa ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa
     3781 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac
     3841 tggctcacct tcgggtgggc ctttctgcgt ttatacctag ggatatattc cgcttcctcg
     3901 gcgaccggtt aaagatcttt gacagctagc tcagtcctag gtataatact agtagacgct
     3961 gattacatgg actgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     4021 aacttgaaaa agtggcaccg agtcggtgct ttttttgacc cgggtggctg ttttggcgga
     4081 tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg tctgataaaa
     4141 cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa
     4201 gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc
     4261 caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     4321 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc
     4381 gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat
     4441 taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc ggatcccatg
     4501 gtacgcgttg attctcctta cgcatctgtg cggtatttca caccgcatgt actagttaac
     4561 ttcaggacct gcagc
//
