LOCUS       sequence-454068-        6689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 acceptor, Position 3 containing 
            CBS4-miniDFR::mCherry2-HyP5SM-LacZ::nosT..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6689)
  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 6689)
  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: pCHW91069
FEATURES             Location/Qualifiers
     source          1..6689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      9..261
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    309..333
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(489..1637)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(1973..2561)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2053..2072)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2732..3523,3524..3592))
                     /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     3355..3374
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3593..3697)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3784..4415
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4507..4531
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    6175..6196
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6211..6216,6217..6234,6235..6241)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6249..6265
                     /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     6254..6276
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6273..6289
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6273..6289
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6285..6608
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    6302..6358
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(6359..6376)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6359..6375)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6368..6390)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6584..6603)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtcaagcaga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg
       61 gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca
      121 tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca
      181 tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg
      241 tgtcatctat gttactagat cgacgcttta cttgtcttct gcacgaagtg gtttaaacta
      301 tcagtgtttg acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat
      361 aatcggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc
      421 caaccacagg gttccccaga tcaggcgctg gctgctgaac ccccagccgg aactgacccc
      481 acaaggccct agcgtttgca atgcaccagg tcatcattga cccaggcgtg ttccaccagg
      541 ccgctgcctc gcaactcttc gcaggcttcg ccgacctgct cgcgccactt cttcacgcgg
      601 gtggaatccg atccgcacat gaggcggaag gtttccagct tgagcgggta cggctcccgg
      661 tgcgagctga aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg gtacttctcc
      721 catatgaatt tcgtgtagtg gtcgccagca aacagcacga cgatttcctc gtcgatcagg
      781 acctggcaac gggacgtttt cttgccacgg tccaggacgc ggaagcggtg cagcagcgac
      841 accgattcca ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc ctgtaggcgc
      901 gacaggcatt cctcggcctt cgtgtaatac cggccattga tcgaccagcc caggtcctgg
      961 caaagctcgt agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt cgcgtactcc
     1021 aacacctgct gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc ggtgtaggtg
     1081 atcttcacgt ccttgttgac gtggaaaatg accttgtttt gcagcgcctc gcgcgggatt
     1141 ttcttgttgc gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat cgctcgcatc
     1201 gtgtccggcc acggcgcaat atcgaacaag gaaagctgca tttccttgat ctgctgcttc
     1261 gtgtgtttca gcaacgcggc ctgcttggcc tcgctgacct gttttgccag gtcctcgccg
     1321 gcggtttttc gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat cgacttcgcc
     1381 aaacctgccg cctcctgttc aagacgacgc gaacgctcca cggcggccga tggcgcgggc
     1441 agggcagggg gagccagttg cacgctgtcg cgctcgatct tggccgtagc ttgctggacc
     1501 atcgagccga cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg gcttgcgatg
     1561 gtttcggcat cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta tgccttccgg
     1621 tcaaacgtcc gattcattca ccctccttgc gggattgccc cgactcacgc cggggcaatg
     1681 tgcccttatt cctgatttga cccgcctggt gccttggtgt ccagataatc caccttatcg
     1741 gcaatgaagt cggtcccgta gaccgtctgg ccgtccttct cgtacttggt attccgaatc
     1801 ttgccctgca cgaataccag cgaccccttg cccaaatact tgccgtgggc ctcggcctga
     1861 gagccaaaac acttgatgcg gaagaagtcg gtgcgctcct gcttgtcgcc ggcatcgttg
     1921 cgccacatct aggatctgcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1981 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2041 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2101 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2161 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2221 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2281 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2341 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2401 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2461 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2521 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2581 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2641 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2701 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     2761 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     2821 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagaaccac
     2881 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     2941 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3001 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3061 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3121 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3181 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3241 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3301 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3361 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3421 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3481 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3541 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3601 tttttcaata ttattgaagc atttatcaag gttattgtct catgagcgga tacatatttg
     3661 aatgtattta gaaaaataaa caaatagggg ttccgcgcac gaattggcca gcgctgccat
     3721 ttttggggtg aggccgttcg cggccgaggg gcgcagcccc tggggggatg ggaggcccgc
     3781 gttagcgggc cgggagggtt cgagaagggg gggcaccccc cttcggcgtg cgcggtcacg
     3841 cgcacagggc gcagccctgg ttaaaaacaa ggtttataaa tattggttta aaagcaggtt
     3901 aaaagacagg ttagcggtgg ccgaaaaacg ggcggaaacc cttgcaaatg ctggattttc
     3961 tgcctgtgga cagcccctca aatgtcaata ggtgcgcccc tcatctgtca gcactctgcc
     4021 cctcaagtgt caaggatcgc gcccctcatc tgtcagtagt cgcgcccctc aagtgtcaat
     4081 accgcagggc acttatcccc aggcttgtcc acatcatctg tgggaaactc gcgtaaaatc
     4141 aggcgttttc gccgatttgc gaggctggcc agctccacgt cgccggccga aatcgagcct
     4201 gcccctcatc tgtcaacgcc gcgccgggtg agtcggcccc tcaagtgtca acgtccgccc
     4261 ctcatctgtc agtgagggcc aagttttccg cgaggtatcc acaacgccgg cggccgcggt
     4321 gtctcgcaca cggcttcgac ggcgtttctg gcgcgtttgc agggccatag acggccgcca
     4381 gcccagcggc gagggcaacc agcccggtga gcgtcgcaaa ggagatcctg atctgactga
     4441 tgggctgcct gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct
     4501 ggctggtggc aggatatatt gtggtgtaaa caaattgacg cttagacaac ttaataacac
     4561 attgcggacg tttttaatgt actggggtgg atgcagtggg ccccactctg tgaagacaaa
     4621 ctagaattcg agctcggaga gtggtcttgg gagagcgatg cgtcttttcc gatgaaccgt
     4681 tccagcaaaa aagactaagc gatgcgtctt ttccgctgaa ccgttccagc aaaaaagact
     4741 aagcgatgcg tcttttccgc tgaaccgttc cagcaaaaaa gactaggaga gcgatgcgtc
     4801 ttttccgctg aaccgttcca gcaaaaaaga ctatcccgtc ctaatacctc tacgtgcaag
     4861 cagtataaaa acaaaataca taaacataac taaaatctca tatcgttgta attgtcatat
     4921 actagtggta attaagtcca gttacactta aggttaaatt tgctgactct tttttacctt
     4981 tcatttctga aaccatggtg agtaaaggag aagaaaacaa cttagctatc attaaagagt
     5041 tcatgcgctt caaagttcac atggagggtt ctgttaacgg tcacgagttc gagatcgaag
     5101 gcgaaggcga gggccgtccg tatgaaggca cccagaccgc caaactgaaa gtgactaaag
     5161 gcggcccgct gccttttgcg tgggacatcc tgagcccgca atttatgtac ggttctaaag
     5221 cgtatgttaa acacccagcg gatatcccgg actatctgaa gctgtctttt ccggaaggtt
     5281 tcaactggga ggtagattta tgcatcctct tgtcatgaga agtcgaattg ttcccattct
     5341 gtgtgttgca gctacagatg gagatacata gagatactcg tggattttgc ttagtgttga
     5401 gttttgttct ggttgtgaac taaaagttta tacatttgca ggaaataaat agccttttgt
     5461 ttaaatcaaa aggtcttacc tatgttagtg tgaagcattg gatcccaaag agagaactcc
     5521 aaaatgcgat gaggcatatt taatcttgtc tggactagta acaggttggg atgaccacct
     5581 gtgaagctcc aacaggattg cctcctcacg caatgtttga ggtctgatgt tcaatagctt
     5641 gttttgtttc actttgcttt ggactttctt ttcgccaatg agctatgttt ctgatggttt
     5701 tcactctttt ggtgtgtaga gagtaatgaa ttttgaagat ggtggtgtcg tgaccgtcac
     5761 tcaggactcc tccctgcagg atggcgagtt catctataaa gttaaactgc gtggtactaa
     5821 ttttccatct gatggcccgg tgatgcagtg taggacgatg ggttgggagg cgtctaccga
     5881 acgcatgtat ccggaagatg gtgcgctgaa aggcgaaatt aaacagcgcc tgaaactgaa
     5941 agatggcggc cattatgacg ctgaagtgaa aaccacgtac aaagccaaga aacctgtgca
     6001 gctgcctggc gcgtacaatg tggatattaa actggacatc ttatctcata atgaagatta
     6061 tacgatcgta gagcaatatg agcgcgcgga gggtcgtcat tctaccggtg gcatggatga
     6121 actatacaaa atgtgagacc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     6181 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     6241 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     6301 caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc tcgaattcac
     6361 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     6421 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     6481 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
     6541 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     6601 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     6661 gtctgctccc ggcatccggg tctcagctt
//
