LOCUS       sequence-324776-        2481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  orthogonal T7 RNAP sfGFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2481)
  AUTHORS   Jung JK, Rasor BJ, Rybnicky GA, Silverman AD, Standeven J, Kuhn R, 
            Granito T, Ekas HM, Wang BM, Karim AS, Lucks JB, Jewett MC
  TITLE     At-Home, Cell-Free Synthetic Biology Education Modules for 
            Transcriptional Regulation and Environmental Water Quality 
            Monitoring.
  JOURNAL   ACS Synth Biol. 2023 Oct 20;12(10):2909-2921. doi: 
            10.1021/acssynbio.3c00223. Epub 2023 Sep 12.
  PUBMED    37699423
REFERENCE   2  (bases 1 to 2481)
  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"; doi: 
            "10.1021/acssynbio.3c00223"; journalName: "ACS Synth Biol"; date: 
            "2023-10-20- 20"; volume: "12"; issue: "10"; pages: "2909-2921"
COMMENT     Sequence Label: pJBL7056
FEATURES             Location/Qualifiers
     source          1..2481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     506..525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             684..704
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(714..716,717..1430)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             714..1427
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(744..772)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1367..1387
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1482..1500)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1496..1543
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1561..2376)
                     /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"
     primer_bind     2284..2303
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2368..2387
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
       61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      601 ggaaaaacgc cagcaacgcg atcccgcgaa attaatacct gacactatag ggagaccaca
      661 acggtttccc tctagaaata attttgttta actttaagaa ggaggaagga tctatgagca
      721 aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta
      781 atgggcacaa attttctgtc cgtggagagg gtgaaggtga tgctacaaac ggaaaactca
      841 cccttaaatt tatttgcact actggaaaac tacctgttcc gtggccaaca cttgtcacta
      901 ctctgaccta tggtgttcaa tgcttttccc gttatccgga tcacatgaaa cggcatgact
      961 ttttcaagag tgccatgccc gaaggttatg tacaggaacg cactatatct ttcaaagatg
     1021 acgggaccta caagacgcgt gctgaagtca agtttgaagg tgataccctt gttaatcgta
     1081 tcgagttaaa gggtattgat tttaaagaag atggaaacat tcttggacac aaactcgagt
     1141 acaactttaa ctcacacaat gtatacatca cggcagacaa acaaaagaat ggaatcaaag
     1201 ctaacttcaa aattcgccac aacgttgaag atggttccgt tcaactagca gaccattatc
     1261 aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcga
     1321 cacaatctgt cctttcgaaa gatcccaacg aaaagcgtga ccacatggtc cttcttgagt
     1381 ttgtaactgc tgctgggatt acacatggca tggatgagct ctacaaataa gtcgaccggc
     1441 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1501 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag ccaattctga
     1561 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1621 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1681 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     1741 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     1801 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
     1861 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     1921 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     1981 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     2041 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     2101 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     2161 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     2221 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     2281 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     2341 cttcgagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     2401 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca
     2461 gagattttga gacacaacgt g
//
