LOCUS       sequence-490750-        7321 bp    DNA     circular SYN 27-OCT-2025
DEFINITION  CaMV 35S-SynPro-12 engineered promoter driving the expression of 
            Renilla luciferase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7321)
  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 7321)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 27, 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: LLP977
FEATURES             Location/Qualifiers
     source          1..7321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1050..1098
                     /note="synthetic polyadenylation signal"
     misc_feature    1112..1203
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     promoter        1217..1396
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     complement(1473..1491)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     intron          1491..1679
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     terminator      3255..3507
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     polyA_signal    3508..3556
                     /note="synthetic polyadenylation signal"
     misc_feature    3570..3661
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     promoter        complement(3680..3698)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3719..3735)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3719..3735)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3732..3754)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3743..3759
                     /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(3767..3773,3774..3791,3792..3797))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3812..3833
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3950..3967)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4121..4709)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4201..4220)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4880..5671,5672..5740))
                     /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     5503..5522
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5741..5845)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5871..6326)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6008..6029)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6220..6239
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6453..6475
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6467..6484
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6468..6484
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6491..6510
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6491..6509
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          6951..7139
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
ORIGIN
        1 taggtgtatt atcccagatc ttatcggaat gggaaagtct ggtaaatctg gaaatggttc
       61 ttacagactt ttggatcatt ataagtacct cacagcttgg ttcgaacttc tcaacttacc
      121 taagaagatt attttcgttg gacacgattg gggtgcttgc ttggcattcc attactcata
      181 cgaacaccaa gataagatca aagctatagt tcatgcagag agtgttgtgg atgtgataga
      241 atcttgggat gagtggccag atattgaaga ggatattgct ttgatcaagt ctgaagaggg
      301 agaaaagatg gttctcgaaa ataacttttt cgtggaaact atgcttcctt caaagattat
      361 gagaaaattg gaaccagaag agtttgctgc atatctcgaa cctttcaagg aaaaaggaga
      421 ggttagaagg ccaacactca gttggcctag ggaaatacca ttagttaagg gaggtaaacc
      481 tgatgttgtg cagatcgtga gaaattataa cgcttacctt agggcatcag atgatttgcc
      541 taagatgttc atcgagtcag atccaggatt tttcagtaat gctatagttg aaggtgcaaa
      601 gaaatttcca aacaccgagt tcgttaaggt gaaaggattg catttctcac aagaagatgc
      661 tcctgatgag atgggaaagt atattaagtc ttttgttgaa agagtgctca aaaacgagca
      721 gggttctcga tctgatgctt ctgaagtaac gtcaacaagc tcccagtctg aggtatgcac
      781 agtggaaacg ccaggttgtg ttggttctta aggtacccgg ccatgctaga gtccgcaaaa
      841 atcaccagtc tctctctaca aatctatctc tctctatttt tctccagaat aatgtgtgag
      901 tagttcccag ataagggaat tagggttctt atagggtttc gctcatgtgt tgagcatata
      961 agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc
     1021 ctaaaaccaa aatccagtga cctactagta ataaaatatc tttattttca ttacatctgt
     1081 gtgttggttt tttgtgtgaa tcgatagtac taacatacgc tctccatcaa aacaaaacga
     1141 aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag tgcaggtgcc agaacatttc
     1201 tctagcgctc tctagagaac cgcaacgttg aaggagccac tgagccgcgg gtttctggag
     1261 tttaatgagc taagcacata cgtcagaaac cattattgcg cgttcaaaag tcgcctaagg
     1321 tcactatcag ctagcaaata tttcttgtca aaaatgctcc actgacgttc cataaattcc
     1381 cctcggtatc caattagagt ccatggatgg aagacgccaa aaacataaag aaaggcccgg
     1441 cgccattcta tccgctggaa gatggaaccg ctggagagca actgcataag gtaagtttct
     1501 gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca
     1561 aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa
     1621 gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcagg
     1681 ctatgaagag atacgccctg gttcctggaa caattgcttt tacagatgca catatcgagg
     1741 tggacatcac ttacgctgag tacttcgaaa tgtccgttcg gttggcagaa gctatgaaac
     1801 gatatgggct gaatacaaat cacagaatcg tcgtatgcag tgaaaactct cttcaattct
     1861 ttatgccggt gttgggcgcg ttatttatcg gagttgcagt tgcgcccgcg aacgacattt
     1921 ataatgaacg tgaattgctc aacagtatgg gcatttcgca gcctaccgtg gtgttcgttt
     1981 ccaaaaaggg gttgcaaaaa attttgaacg tgcaaaaaaa gctcccaatc atccaaaaaa
     2041 ttattatcat ggattctaaa acggattacc agggatttca gtcgatgtac acgttcgtca
     2101 catctcatct acctcccggt tttaatgaat acgattttgt gccagagtcc ttcgataggg
     2161 acaagacaat tgcactgatc atgaactcct ctggatctac tggtctgcct aaaggtgtcg
     2221 ctctgcctca tagaactgcc tgcgtgagat tctcgcatgc cagagatcct atttttggca
     2281 atcaaatcat tccggatact gcgattttaa gtgttgttcc attccatcac ggttttggaa
     2341 tgtttactac actcggatat ttgatatgtg gatttcgagt cgtcttaatg tatagatttg
     2401 aagaagagct gtttctgagg agccttcagg attacaagat tcaaagtgcg ctgctggtgc
     2461 caaccctatt ctccttcttc gccaaaagca ctctgattga caaatacgat ttatctaatt
     2521 tacacgaaat tgcttctggt ggcgctcccc tctctaagga agtcggggaa gcggttgcca
     2581 agaggttcca tctgccaggt atcaggcaag gatatgggct cactgagact acatcagcta
     2641 ttctgattac acccgagggg gatgataaac cgggcgcggt cggtaaagtt gttccatttt
     2701 ttgaagcgaa ggttgtggat ctggataccg ggaaaacgct gggcgttaat caaagaggcg
     2761 aactgtgtgt gagaggtcct atgattatgt ccggttatgt aaacaatccg gaagcgacca
     2821 acgccttgat tgacaaggat ggatggctac attctggaga catagcttac tgggacgaag
     2881 acgaacactt cttcatcgtt gaccgcctga agtctctgat taagtacaaa ggctatcagg
     2941 tggctcccgc tgaattggaa tccatcttgc tccaacaccc caacatcttc gacgcaggtg
     3001 tcgcaggtct tcccgacgat gacgccggtg aacttcccgc cgccgttgtt gttttggagc
     3061 acggaaagac gatgacggaa aaagagatcg tggattacgt cgccagtcaa gtaacaaccg
     3121 cgaaaaagtt gcgcggagga gttgtgtttg tggacgaagt accgaaaggt cttaccggaa
     3181 aactcgacgc aagaaaaatc agagagatcc tcataaaggc caagaagggc ggaaagatcg
     3241 ccgtgtaact cgaggatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg
     3301 ttgccggtct tgcgacgatt atcatataat ttctgttgaa ttacgttaag catgtaataa
     3361 ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat
     3421 tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc
     3481 gcgcggtgtc atctatgtta ctagatcaat aaaatatctt tattttcatt acatctgtgt
     3541 gttggttttt tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa
     3601 caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc
     3661 tgagctccag cttttgttcc ctttagtgag ggttaatttc gagcttggcg taatcatggt
     3721 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3781 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3841 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3901 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3961 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4021 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4081 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4141 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4201 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4261 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4321 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4381 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4441 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4501 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4561 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4621 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4681 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4741 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4801 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4861 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4921 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4981 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5041 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5101 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5161 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5221 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5281 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5341 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5401 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5461 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5521 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5581 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5641 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5701 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5761 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5821 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     5881 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5941 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     6001 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     6061 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     6121 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     6181 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     6241 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     6301 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     6361 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     6421 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     6481 cagtgaattg taatacgact cactataggg cgaattgggt acaagcttga tcatgccgaa
     6541 ttccgaacgc gtttgagact tttcaacaaa gggtaatatc gggaaacctc ctcggattcc
     6601 attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca
     6661 aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc
     6721 ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt
     6781 cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc
     6841 acggaacgtg attgaataac tttggtatat aatctacccg agactcaaac ggtgggacgg
     6901 atccgtcgac ataactgcag atgacttcaa aggtgtatga tcctgagcag gtaagtttct
     6961 gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca
     7021 aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa
     7081 gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcaga
     7141 gaaaaagaat gataactggt ccacaatggt gggcaagatg taaacagatg aacgtgcttg
     7201 attctttcat taattactac gattcagaaa agcacgctga gaatgcagtt atctttcttc
     7261 acggaaacgc tgcatcttca tatttgtgga gacatgttgt gcctcacatt gaaccagtgg
     7321 c
//
