LOCUS       sequence-454090-        6620 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 acceptor, Position 3 containing 
            CBS4-CMV1::LoxP-mCherry-stop-LacZ::nosT..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6620)
  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 6620)
  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: pCHW111118
FEATURES             Location/Qualifiers
     source          1..6620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(36..624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(116..135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(795..1586,1587..1655))
                     /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     1418..1437
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1656..1760)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1847..2478
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    2570..2594
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    complement(3008..3041)
                     /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(3268..3286)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3895..4069
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     protein_bind    complement(4079..4112)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4169..4190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4205..4210,4211..4228,4229..4235)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4243..4259
                     /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)."
     primer_bind     4248..4270
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4267..4283
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4267..4283
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4279..4602
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    4296..4352
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4353..4370)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4353..4369)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4362..4384)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4578..4597)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     terminator      4692..4944
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    4992..5016
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(5172..6320)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
ORIGIN
        1 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
       61 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
      121 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
      181 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
      241 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
      301 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
      361 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
      421 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
      481 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
      541 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
      601 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
      661 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
      721 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
      781 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
      841 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
      901 ttaccatctg gccccagtgc tgcaatgata ccgcgagaac cacgctcacc ggctccagat
      961 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     1021 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     1081 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     1141 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     1201 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     1261 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     1321 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     1381 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     1441 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     1501 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     1561 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     1621 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     1681 agcatttatc aaggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     1741 aaacaaatag gggttccgcg cacgaattgg ccagcgctgc catttttggg gtgaggccgt
     1801 tcgcggccga ggggcgcagc ccctgggggg atgggaggcc cgcgttagcg ggccgggagg
     1861 gttcgagaag ggggggcacc ccccttcggc gtgcgcggtc acgcgcacag ggcgcagccc
     1921 tggttaaaaa caaggtttat aaatattggt ttaaaagcag gttaaaagac aggttagcgg
     1981 tggccgaaaa acgggcggaa acccttgcaa atgctggatt ttctgcctgt ggacagcccc
     2041 tcaaatgtca ataggtgcgc ccctcatctg tcagcactct gcccctcaag tgtcaaggat
     2101 cgcgcccctc atctgtcagt agtcgcgccc ctcaagtgtc aataccgcag ggcacttatc
     2161 cccaggcttg tccacatcat ctgtgggaaa ctcgcgtaaa atcaggcgtt ttcgccgatt
     2221 tgcgaggctg gccagctcca cgtcgccggc cgaaatcgag cctgcccctc atctgtcaac
     2281 gccgcgccgg gtgagtcggc ccctcaagtg tcaacgtccg cccctcatct gtcagtgagg
     2341 gccaagtttt ccgcgaggta tccacaacgc cggcggccgc ggtgtctcgc acacggcttc
     2401 gacggcgttt ctggcgcgtt tgcagggcca tagacggccg ccagcccagc ggcgagggca
     2461 accagcccgg tgagcgtcgc aaaggagatc ctgatctgac tgatgggctg cctgtatcga
     2521 gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat
     2581 attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa
     2641 tgtactgggg tggatgcagt gggccccact ctgtgaagac aaactagaat tcgagctcgg
     2701 agagtggtct tgggagagcg atgcgtcttt tccgatgaac cgttccagca aaaaagacta
     2761 agcgatgcgt cttttccgct gaaccgttcc agcaaaaaag actaagcgat gcgtcttttc
     2821 cgctgaaccg ttccagcaaa aaagactagg agagcgatgc gtcttttccg ctgaaccgtt
     2881 ccagcaaaaa agactatccc tactgtttat ttacaagagc gtacggttca atccctgcct
     2941 cccctgtaaa actacccttt gaaaacctct ctttcttaat cttttctttg taattcccca
     3001 tgacctaata acttcgtata gcatacatta tacgaagtta tattaagggt tgaatgaaaa
     3061 ctaatctttt tctctttctc atcttttcac ttctcctatc attatcctcg gccgaattcg
     3121 gaggtgtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     3181 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     3241 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     3301 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     3361 accccgccga catccccgac tacttgaagc tgtccttccc cgagtaatag tgaggcttca
     3421 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     3481 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     3541 acggccccgt aatgcagaag aaaaccatgg gctgggaggc ctcctccgag cggatgtacc
     3601 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     3661 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     3721 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     3781 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg
     3841 gtggccatga tgagctttaa gcttctctag ctagagtcga tcgacaagct cgagtttctc
     3901 cataataatg tgtgagtagt tcccagataa gggaattagg gttcctatag ggtttcgctc
     3961 atgtgttgag catataagaa acccttagta tgtatttgta tttgtaaaat acttctatca
     4021 ataaaatttc taattcctaa aaccaaaatc cagtactaaa atccagatcg ctgacctaat
     4081 aacttcgtat agcatacatt atacgaagtt atattaaggg ttgccattga gaccccgact
     4141 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     4201 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     4261 ttcacacagg aaacagctat gaccatgatt acgccaagct tgcatgcctg caggtcgact
     4321 ctagaggatc cccgggtacc gagctcgaat tcactggccg tcgttttaca acgtcgtgac
     4381 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     4441 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     4501 ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4561 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4621 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgggtctcag
     4681 cttgtcaagc agatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg
     4741 ccggtcttgc gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta
     4801 acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat
     4861 acatttaata cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg
     4921 cggtgtcatc tatgttacta gatcgacgct ttacttgtct tctgcacgaa gtggtttaaa
     4981 ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag
     5041 aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca
     5101 tgccaaccac agggttcccc agatcaggcg ctggctgctg aacccccagc cggaactgac
     5161 cccacaaggc cctagcgttt gcaatgcacc aggtcatcat tgacccaggc gtgttccacc
     5221 aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct gctcgcgcca cttcttcacg
     5281 cgggtggaat ccgatccgca catgaggcgg aaggtttcca gcttgagcgg gtacggctcc
     5341 cggtgcgagc tgaaatagtc gaacatccgt cgggccgtcg gcgacagctt gcggtacttc
     5401 tcccatatga atttcgtgta gtggtcgcca gcaaacagca cgacgatttc ctcgtcgatc
     5461 aggacctggc aacgggacgt tttcttgcca cggtccagga cgcggaagcg gtgcagcagc
     5521 gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc ccatcgccgt cgcctgtagg
     5581 cgcgacaggc attcctcggc cttcgtgtaa taccggccat tgatcgacca gcccaggtcc
     5641 tggcaaagct cgtagaacgt gaaggtgatc ggctcgccga taggggtgcg cttcgcgtac
     5701 tccaacacct gctgccacac cagttcgtca tcgtcggccc gcagctcgac gccggtgtag
     5761 gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc ctcgcgcggg
     5821 attttcttgt tgcgcgtggt gaacagggca gagcgggccg tgtcgtttgg catcgctcgc
     5881 atcgtgtccg gccacggcgc aatatcgaac aaggaaagct gcatttcctt gatctgctgc
     5941 ttcgtgtgtt tcagcaacgc ggcctgcttg gcctcgctga cctgttttgc caggtcctcg
     6001 ccggcggttt ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt catcgacttc
     6061 gccaaacctg ccgcctcctg ttcaagacga cgcgaacgct ccacggcggc cgatggcgcg
     6121 ggcagggcag ggggagccag ttgcacgctg tcgcgctcga tcttggccgt agcttgctgg
     6181 accatcgagc cgacggactg gaaggtttcg cggggcgcac gcatgacggt gcggcttgcg
     6241 atggtttcgg catcctcggc ggaaaacccc gcgtcgatca gttcttgcct gtatgccttc
     6301 cggtcaaacg tccgattcat tcaccctcct tgcgggattg ccccgactca cgccggggca
     6361 atgtgccctt attcctgatt tgacccgcct ggtgccttgg tgtccagata atccacctta
     6421 tcggcaatga agtcggtccc gtagaccgtc tggccgtcct tctcgtactt ggtattccga
     6481 atcttgccct gcacgaatac cagcgacccc ttgcccaaat acttgccgtg ggcctcggcc
     6541 tgagagccaa aacacttgat gcggaagaag tcggtgcgct cctgcttgtc gccggcatcg
     6601 ttgcgccaca tctaggatct
//
