LOCUS       sequence-490465-        7318 bp    DNA     circular SYN 23-OCT-2025
DEFINITION  CaMV 35S-SynPro-14 engineered promoter driving the expression of 
            Renilla luciferase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7318)
  AUTHORS   Khan MA, Herring G, Zhu JY, Oliva M, Fourie E, Johnston B, Zhang Z, 
            Potter J, Pineda L, Pflueger J, Swain T, Pflueger C, Lloyd JPB, 
            Secco D, Small I, Kidd BN, Lister R
  TITLE     CRISPRi-based circuits to control gene expression in plants.
  JOURNAL   Nat Biotechnol. 2025 Mar;43(3):416-430. doi: 
            10.1038/s41587-024-02236-w. Epub 2024 May 20.
  PUBMED    38769424
REFERENCE   2  (bases 1 to 7318)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41587-024-02236-w"; journalName: "Nat Biotechnol"; date: 
            "2025-03"; volume: "43"; issue: "3"; pages: "416-430"
COMMENT     Sequence Label: LLP979
FEATURES             Location/Qualifiers
     source          1..7318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1083..1271
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     polyA_signal    2503..2551
                     /note="synthetic polyadenylation signal"
     misc_feature    2565..2656
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     promoter        2670..2849
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     complement(2926..2944)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     intron          2944..3132
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     terminator      4708..4960
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     polyA_signal    4961..5009
                     /note="synthetic polyadenylation signal"
     misc_feature    5023..5114
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     promoter        complement(5133..5151)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5172..5188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5172..5188)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5185..5207)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5196..5212
                     /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(5220..5226,5227..5244,5245..5250))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5265..5286
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5403..5420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5574..6162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5654..5673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6333..7124,7125..7193))
                     /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     6956..6975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7194..7298)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtacaag
      661 cttgatcatg ccgaattccg aacgcgtttg agacttttca acaaagggta atatcgggaa
      721 acctcctcgg attccattgc ccagctatct gtcacttcat caaaaggaca gtagaaaagg
      781 aaggtggcac ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt caagatgcct
      841 ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag
      901 acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact gacgtaaggg
      961 atgacgcaca atcccacgtg gtaacttgct ccatgtctgg tatataactt tacgtatagg
     1021 tttagagtgg ggatccgtcg acataactgc agatgacttc aaaggtgtat gatcctgagc
     1081 aggtaagttt ctgcttctac ctttgatata tatataataa ttatcattaa ttagtagtaa
     1141 tataatattt caaatatttt tttcaaaata aaagaatgta gtatatagca attgcttttc
     1201 tgtagtttat aagtgtgtat attttaattt ataacttttc taatatatga ccaaaatttg
     1261 ttgatgtgca gagaaaaaga atgataactg gtccacaatg gtgggcaaga tgtaaacaga
     1321 tgaacgtgct tgattctttc attaattact acgattcaga aaagcacgct gagaatgcag
     1381 ttatctttct tcacggaaac gctgcatctt catatttgtg gagacatgtt gtgcctcaca
     1441 ttgaaccagt ggctaggtgt attatcccag atcttatcgg aatgggaaag tctggtaaat
     1501 ctggaaatgg ttcttacaga cttttggatc attataagta cctcacagct tggttcgaac
     1561 ttctcaactt acctaagaag attattttcg ttggacacga ttggggtgct tgcttggcat
     1621 tccattactc atacgaacac caagataaga tcaaagctat agttcatgca gagagtgttg
     1681 tggatgtgat agaatcttgg gatgagtggc cagatattga agaggatatt gctttgatca
     1741 agtctgaaga gggagaaaag atggttctcg aaaataactt tttcgtggaa actatgcttc
     1801 cttcaaagat tatgagaaaa ttggaaccag aagagtttgc tgcatatctc gaacctttca
     1861 aggaaaaagg agaggttaga aggccaacac tcagttggcc tagggaaata ccattagtta
     1921 agggaggtaa acctgatgtt gtgcagatcg tgagaaatta taacgcttac cttagggcat
     1981 cagatgattt gcctaagatg ttcatcgagt cagatccagg atttttcagt aatgctatag
     2041 ttgaaggtgc aaagaaattt ccaaacaccg agttcgttaa ggtgaaagga ttgcatttct
     2101 cacaagaaga tgctcctgat gagatgggaa agtatattaa gtcttttgtt gaaagagtgc
     2161 tcaaaaacga gcagggttct cgatctgatg cttctgaagt aacgtcaaca agctcccagt
     2221 ctgaggtatg cacagtggaa acgccaggtt gtgttggttc ttaaggtacc cggccatgct
     2281 agagtccgca aaaatcacca gtctctctct acaaatctat ctctctctat ttttctccag
     2341 aataatgtgt gagtagttcc cagataaggg aattagggtt cttatagggt ttcgctcatg
     2401 tgttgagcat ataagaaacc cttagtatgt atttgtattt gtaaaatact tctatcaata
     2461 aaatttctaa ttcctaaaac caaaatccag tgacctacta gtaataaaat atctttattt
     2521 tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag tactaacata cgctctccat
     2581 caaaacaaaa cgaaacaaaa caaactagca aaataggctg tccccagtgc aagtgcaggt
     2641 gccagaacat ttctctagcg ctctctagag aaccgcaacg ttgaaggagc cactgagccg
     2701 cgggtttctg gagtttaatg agctaagcac atacgtcaga aaccattatt gcgcgttcaa
     2761 aagtcgccta aggtcactat cagctagcaa atatttcttg tcaaaaatgc tccactgacg
     2821 ttccataaat tcccctcggt atccaattag agtccatgga tggaagacgc caaaaacata
     2881 aagaaaggcc cggcgccatt ctatccgctg gaagatggaa ccgctggaga gcaactgcat
     2941 aaggtaagtt tctgcttcta cctttgatat atatataata attatcatta attagtagta
     3001 atataatatt tcaaatattt ttttcaaaat aaaagaatgt agtatatagc aattgctttt
     3061 ctgtagttta taagtgtgta tattttaatt tataactttt ctaatatatg accaaaattt
     3121 gttgatgtgc aggctatgaa gagatacgcc ctggttcctg gaacaattgc ttttacagat
     3181 gcacatatcg aggtggacat cacttacgct gagtacttcg aaatgtccgt tcggttggca
     3241 gaagctatga aacgatatgg gctgaataca aatcacagaa tcgtcgtatg cagtgaaaac
     3301 tctcttcaat tctttatgcc ggtgttgggc gcgttattta tcggagttgc agttgcgccc
     3361 gcgaacgaca tttataatga acgtgaattg ctcaacagta tgggcatttc gcagcctacc
     3421 gtggtgttcg tttccaaaaa ggggttgcaa aaaattttga acgtgcaaaa aaagctccca
     3481 atcatccaaa aaattattat catggattct aaaacggatt accagggatt tcagtcgatg
     3541 tacacgttcg tcacatctca tctacctccc ggttttaatg aatacgattt tgtgccagag
     3601 tccttcgata gggacaagac aattgcactg atcatgaact cctctggatc tactggtctg
     3661 cctaaaggtg tcgctctgcc tcatagaact gcctgcgtga gattctcgca tgccagagat
     3721 cctatttttg gcaatcaaat cattccggat actgcgattt taagtgttgt tccattccat
     3781 cacggttttg gaatgtttac tacactcgga tatttgatat gtggatttcg agtcgtctta
     3841 atgtatagat ttgaagaaga gctgtttctg aggagccttc aggattacaa gattcaaagt
     3901 gcgctgctgg tgccaaccct attctccttc ttcgccaaaa gcactctgat tgacaaatac
     3961 gatttatcta atttacacga aattgcttct ggtggcgctc ccctctctaa ggaagtcggg
     4021 gaagcggttg ccaagaggtt ccatctgcca ggtatcaggc aaggatatgg gctcactgag
     4081 actacatcag ctattctgat tacacccgag ggggatgata aaccgggcgc ggtcggtaaa
     4141 gttgttccat tttttgaagc gaaggttgtg gatctggata ccgggaaaac gctgggcgtt
     4201 aatcaaagag gcgaactgtg tgtgagaggt cctatgatta tgtccggtta tgtaaacaat
     4261 ccggaagcga ccaacgcctt gattgacaag gatggatggc tacattctgg agacatagct
     4321 tactgggacg aagacgaaca cttcttcatc gttgaccgcc tgaagtctct gattaagtac
     4381 aaaggctatc aggtggctcc cgctgaattg gaatccatct tgctccaaca ccccaacatc
     4441 ttcgacgcag gtgtcgcagg tcttcccgac gatgacgccg gtgaacttcc cgccgccgtt
     4501 gttgttttgg agcacggaaa gacgatgacg gaaaaagaga tcgtggatta cgtcgccagt
     4561 caagtaacaa ccgcgaaaaa gttgcgcgga ggagttgtgt ttgtggacga agtaccgaaa
     4621 ggtcttaccg gaaaactcga cgcaagaaaa atcagagaga tcctcataaa ggccaagaag
     4681 ggcggaaaga tcgccgtgta actcgaggat cgttcaaaca tttggcaata aagtttctta
     4741 agattgaatc ctgttgccgg tcttgcgacg attatcatat aatttctgtt gaattacgtt
     4801 aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt
     4861 agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag
     4921 gataaattat cgcgcgcggt gtcatctatg ttactagatc aataaaatat ctttattttc
     4981 attacatctg tgtgttggtt ttttgtgtga atcgatagta ctaacatacg ctctccatca
     5041 aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc cccagtgcaa gtgcaggtgc
     5101 cagaacattt ctctgagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg
     5161 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5221 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5281 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5341 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5401 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5461 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5521 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5581 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5641 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5701 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5761 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5821 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5881 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5941 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6001 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6061 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6121 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6181 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6241 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6301 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6361 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6421 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6481 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6541 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6601 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6661 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6721 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6781 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6841 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6901 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6961 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7021 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7081 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7141 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7201 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7261 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgc
//
