LOCUS       sequence-304707-        5468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for STARR-seq in tobacco leaves. Enhancer candidates can be 
            inserted into the 3'-UTR of the GFP reporter gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5468)
  AUTHORS   Jores T, Tonnies J, Dorrity MW, Cuperus J, Fields S, Queitsch C
  TITLE     Identification of Plant Enhancers and Their Constituent Elements by 
            STARR-seq in Tobacco Leaves.
  JOURNAL   Plant Cell. 2020 May 14. pii: tpc.20.00155. doi: 
            10.1105/tpc.20.00155.
  PUBMED    32409318
REFERENCE   2  (bases 1 to 5468)
  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: "Plant Cell.
            2020 May 14. pii: tpc.20.00155. doi: 10.1105/tpc.20.00155."
COMMENT     Sequence Label: pPSdown
FEATURES             Location/Qualifiers
     source          1..5468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     70..86
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     71..87
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(157..159,160..873)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(199..220)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(460..479)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     807..828
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1224..1244)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1224..1242)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1263..1279)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1263..1279)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1276..1298)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1287..1303
                     /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(1311..1317,1318..1335,1336..1341))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1356..1377
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1494..1511)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1616..1640
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(1731..2319)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1811..1830)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2490..3278)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNGTLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      3569..4004
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    4132..4154
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     terminator      complement(4178..4430)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(4450..5001)
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     promoter        complement(5043..5222)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     join(5454..5468,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(5468,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg
       61 ggccccccct cgaggtcgac ggtatcgata agcttgatat cgaattccgc ggccgcagga
      121 gctgccgtct tcagtgcctg caggcataga agacccgtga gcaagggcga ggagctgttc
      181 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      241 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      301 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcac ctacggcgtg
      361 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      421 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      481 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
      541 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
      601 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
      661 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
      721 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
      781 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
      841 atcactcacg gcatggacga gctgtacaag taaaactccg agaccacaag gcgcgcctag
      901 tggtctccag gaaaattgcc cagaattaat tccccaactg cacagaacaa atttcccttt
      961 gcttttgtgt agacctcaaa actttatccc ccatctttga tcttatccct tgtttttctg
     1021 cttttttctt ctttcttggg ttttgatttc cggacttaac gtttgttttc cggtttgcga
     1081 gacatattct atcggattct caactgtctg atgaaataaa tatgtaatgt tctataagtc
     1141 tttcaatttg atgggatgtt ccacttctgt ataggagcca attgattgac aacatgcatc
     1201 aatcggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     1261 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     1321 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1381 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1441 atcggccaac gctcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     1501 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1561 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgaa ggccttgaca
     1621 ggatatattg gcgggtaaac taagtcgctg tatgtgtttg tttgagatct catgtgagca
     1681 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1741 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1801 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     1861 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     1921 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     1981 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2041 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2101 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2161 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaga
     2221 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2281 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2341 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt cattagatta
     2401 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2461 agtatatatg tgtaacattg gtctagtgat tatttgccga ctaccttggt gatctcgcct
     2521 ttcacgtagt gaacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttgt
     2581 ccaagataag cctgcctagc ttcaagtatg acgggctgat actgggccgg caggcgctcc
     2641 attgcccagt cggcagcgac atccttcggc gcgattttgc cggttactgc gctgtaccaa
     2701 atgcgggaca acgtaagcac tacatttcgc tcatcgccag cccagtcggg cggcgagttc
     2761 catagcgtta aggttccatt tagcgcctca aatagatcct gttcaggaac cggatcaaag
     2821 agttcctccg ccgctggacc taccaaggca acgctatgtt ctcttgcttt tgtcagcaag
     2881 atagccagat caatgtcgat cgtggctggc tcgaagatac ctgcaagaat gtcattgcgc
     2941 tgccattctc caaattgcag ttcgcgctta gctggataac gccacggaat gatgtcgtcg
     3001 tgcacaacaa tggtgacttc tacagcgcgg agaatctcgc tctctccagg ggaagccgaa
     3061 gtttccaaaa ggtcgttgat caaagctcgc cgcgttgttt catcaagcct tacggtcacc
     3121 gtaaccagca aatcaatatc actgtgtggc ttcaggccgc catccactgc ggagccgtac
     3181 aaatgtacgg ccagcaacgt cggttcgaga tggcgctcga tgacgccaac tacctctgat
     3241 agttgagtcg atacttcggc gatcaccgct tccctcataa caccccttgt attactgttt
     3301 atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc aatgtaacat
     3361 cagagatttt gagacacaac gtggctttgt tgaataaatc gaacttttgc tgagttgaag
     3421 gatcagatca cgcatcttcc cgacaacgca gaccgttccg tggcaaagca aaagttcaaa
     3481 atcaccaact ggtccaccta caacaaagct ctcatcaacc gtggctccct cactttctgg
     3541 ctggatgatg gggcgattca ggcgatcccc atccaacagc ccgccgtcga gcgggctttt
     3601 ttatccccgg aagcctgtgg atagagggta gttatccacg tgaaaccgct aatgccccgc
     3661 aaagccttga ttcacggggc tttccggccc gctccaaaaa ctatccacgt gaaatcgcta
     3721 atcagggtac gtgaaatcgc taatcggagt acgtgaaatc gctaataagg tcacgtgaaa
     3781 tcgctaatca aaaaggcacg tgagaacgct aatagccctt tcagatcaac agcttgcaaa
     3841 cacccctcgc tccggcaagt agttacagca agtagtatgt tcaattagct tttcaattat
     3901 gaatatatat atcaattatt ggtcgccctt ggcttgtgga caatgcgcta cgcgcaccgg
     3961 ctccgcccgt ggacaaccgc aagcggttgc ccaccgtcga gcgcctttgc ccacaacccg
     4021 gcggccggcc gcaacagatc gttttataaa tttttttttt ttgaaaaaga aaaagcccga
     4081 aaggcggcaa cctctcgggc ttctggattt ccgatccccg gaattagatc ttggcaggat
     4141 atattgtggt gtaacgttat cagcttgcat gccggtcgat ctagtaacat agatgacacc
     4201 gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc gtattaaatg
     4261 tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat gcattacatg
     4321 ttaattatta catgcttaac gtaattcaac agaaattata tgataatcat cgcaagaccg
     4381 gcaacaggat tcaatcttaa gaaactttat tgccaaatgt tttaacgatc tgcttgactc
     4441 taggggtcat cagatttcgg tgacgggcag gaccggacgg ggcggcaccg gcaggctgaa
     4501 gtccagctgc cagaaaccca cgtcatgcca gttcccgtgc ttgaagccgg ccgcccgcag
     4561 catgccgcgg ggggcatatc cgagcgcctc gtgcatgcgc acgctcgggt cgttgggcag
     4621 cccgatgaca gcgaccacgc tcttgaagcc ctgtgcctcc agggacttca gcaggtgggt
     4681 gtagagcgtg gagcccagtc ccgtccgctg gtggcggggg gagacgtaca cggtggactc
     4741 ggccgtccag tcgtaggcgt tgcgtgcctt ccagggaccc gcgtaggcga tgccggcgac
     4801 ctcgccgtcc acctcggcga cgagccaggg atagcgctcc cgcagacgga cgaggtcgtc
     4861 cgtccactcc tgcggttcct gcggctcggt acggaagttg accgtgcttg tctcgatgta
     4921 gtggttgacg atggtgcaga ccgccggcat gtccgcctcg gtggcacggc ggatgtcggc
     4981 cgggcgtcgt tctgggctca tggtagatcc cctcgatcga gttgagagtg aatatgagac
     5041 tctaattgga taccgagggg aatttatgga acgtcagtgg agcatttttg acaagaaata
     5101 tttgctagct tatagtgacc ttaggcgact tttgaacgcg caataatggt ttctgacgta
     5161 tgtgcttagc tcattaaact ccagaaaccc gcggctcagt ggctccttca acgttgcggt
     5221 tctgtcagtt ccaaacgtaa aacggcttgt cccgcgtcat cggcgggggt cataacgtga
     5281 ctcccttaat tctccgctca tgatcgataa cattaacgct tacaatttcc attcgccatt
     5341 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     5401 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     5461 acgacgtt
//
