LOCUS       sequence-452569-        3714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0, N-terminal tag module (CCAT-AATG) containing LoxP-mCherry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3714)
  AUTHORS   Chiang BJ, Lin KY, Chen YF, Huang CY, Goh FJ, Huang LT, Chen LH, Wu 
            CH
  TITLE     Development of a tightly regulated copper-inducible transient gene 
            expression system in Nicotiana benthamiana incorporating suicide 
            exon and Cre recombinase
  JOURNAL   bioRxiv 2024.03.23.586378
REFERENCE   2  (bases 1 to 3714)
  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: "bioRxiv 
            2024.03.23.586378"
COMMENT     Sequence Label: pCHW110034
FEATURES             Location/Qualifiers
     source          1..3714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(169..757)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(249..268)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(935..1744)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1658..1677
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1736..1755
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1886..1908)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2008..2027
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2221..2243
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2306..2339)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2424..3128
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2566..2584)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3184..3358
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     protein_bind    complement(3368..3401)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3481..3497)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3481..3497)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3494..3516)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3505..3521
                     /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(3529..3535,3536..3553,3554..3559))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3574..3595
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(join(3712..3714,1..15))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
       61 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
      121 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
      181 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
      241 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
      301 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
      361 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
      421 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
      481 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
      541 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
      601 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
      661 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
      721 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
      781 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
      841 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caagcccaat
      901 ctgaataatg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat
      961 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     1021 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     1081 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     1141 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     1201 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     1261 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     1321 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     1381 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     1441 ttccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     1501 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     1561 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     1621 acaagcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     1681 ataaatcagc atccatgttg gaatttaatc gcggcctcga cgtttcccgt tgaatatggc
     1741 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata
     1801 tatttttatc ttgtgcaatg taacatcaga gattttgaga cacgggccag agctgcatcg
     1861 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     1921 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     1981 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     2041 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
     2101 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     2161 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     2221 cccagtcacg acgttgtaaa acgacggcca gagaattcga gctcggtacc tcgcgaatac
     2281 atctagatgg tctctccatg acctaataac ttcgtatagc atacattata cgaagttata
     2341 ttaagggttg aatgaaaact aatctttttc tctttctcat cttttcactt ctcctatcat
     2401 tatcctcggc cgaattcgga ggtgtgagca agggcgagga ggataacatg gccatcatca
     2461 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     2521 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     2581 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     2641 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     2701 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     2761 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     2821 tcccctccga cggccccgta atgcagaaga aaaccatggg ctgggaggcc tcctccgagc
     2881 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     2941 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     3001 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     3061 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     3121 tgtacaaggg tggccatgat gagctttaag cttctctagc tagagtcgat cgacaagctc
     3181 gagtttctcc ataataatgt gtgagtagtt cccagataag ggaattaggg ttcctatagg
     3241 gtttcgctca tgtgttgagc atataagaaa cccttagtat gtatttgtat ttgtaaaata
     3301 cttctatcaa taaaatttct aattcctaaa accaaaatcc agtactaaaa tccagatcgc
     3361 tgacctaata acttcgtata gcatacatta tacgaagtta tattaagggt tgaatgtgag
     3421 accatcggat cccgggcccg tcgactgcag aggcctgcat gcaagcttgg tgtaatcatg
     3481 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3541 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     3601 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3661 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctc
//
