LOCUS       sequence-267167-        2625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sfGFP driven by a T7 promoter with the tetO sequence 36 bp upstream 
            from the promoter start site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2625)
  AUTHORS   McManus JB, Lux MW, Murray RM, Emanuel PA
  TITLE     A method for cost-effective and rapid characterization of engineered
            T7-Based transcription factors by cell-free protein synthesis 
            reveals insights into the regulation of T7 Rna polymerase-driven 
            expression.
  JOURNAL   Arch Biochem Biophys. 2019 Jul 18. pii: S0003-9861(19)30288-7. doi: 
            10.1016/j.abb.2019.07.010.
  PUBMED    31326518
REFERENCE   2  (bases 1 to 2625)
  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: "Arch 
            Biochem Biophys. 2019 Jul 18. pii: S0003-9861(19)30288-7. doi: 
            10.1016/j.abb.2019.07.010."
COMMENT     Sequence Label: pY71-tet.I.-1
FEATURES             Location/Qualifiers
     source          1..2625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     705..724
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     789..808
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      919..1507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1408..1427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1537..1555
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        1556..1574
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        1607..1656
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             1687..1698
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1708..1728
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2368..2415
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             2416..2439
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(join(2607..2625,1..797))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
       61 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
      121 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
      181 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
      241 aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
      301 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
      361 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
      421 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
      481 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
      541 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
      601 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
      661 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
      721 tacccatata aatcagcatc catgttggaa tttaatcgcg gcttcgagca agacgtttcc
      781 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
      841 gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg
      901 tgagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
      961 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1021 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     1081 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1141 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1201 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1261 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1321 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1381 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1441 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1501 atggaaaaac gccagatctc gatcccgcga gctacgtccc tatcagtgat agagataata
     1561 cgactcacta taggcttacc tcttcagagg tcactgacct gacaagctgt caccggatgt
     1621 gctttccggt ctgatgagtc cgtgaggacg aaacagcctc tacaaataat tttgtttaat
     1681 ctagagaaag aggagaaata ctagatggag aatttatact tccagagtag caaaggtgaa
     1741 gaactgttta ccggcgttgt gccgattctg gtggaactgg atggcgatgt gaacggtcac
     1801 aaattcagcg tgcgtggtga aggtgaaggc gatgccacga ttggcaaact gacgctgaaa
     1861 tttatctgca ccaccggcaa actgccggtg ccgtggccga cgctggtgac caccctgacc
     1921 tatggcgttc agtgttttag tcgctatccg gatcacatga aacgtcacga tttctttaaa
     1981 tctgcaatgc cggaaggcta tgtgcaggaa cgtacgatta gctttaaaga tgatggcaaa
     2041 tataaaacgc gcgccgttgt gaaatttgaa ggcgataccc tggtgaaccg cattgaactg
     2101 aaaggcacgg attttaaaga agatggcaat atcctgggcc ataaactgga atacaacttt
     2161 aatagccata atgtttatat tacggcggat aaacagaaaa atggcatcaa agcgaatttt
     2221 accgttcgcc ataacgttga agatggcagt gtgcagctgg cagatcatta tcagcagaat
     2281 accccgattg gtgatggtcc ggtgctgctg ccggataatc attatctgag cacgcagacc
     2341 gttctgtcta aagatccgaa cgaaaaacgg gaccacatgg ttctgcacga atatgtgaat
     2401 gcggcaggta ttacgtggag ccatccgcag ttcgaaaaat aataaacaat aactgaatag
     2461 ggatcccgac tggcgagagc caggtaacga atggatcctt cagccaaaaa acttaagacc
     2521 gccggtcttg tccactacct tgcagtaatg cggtggacag gatcggcggt tttcttttct
     2581 cttctcaagc tgaaagccaa ttctgattag aaaaactcat cgagc
//
