LOCUS       sequence-441001-        4348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S2TAG/E17TAG GFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4348)
  AUTHORS   Pirman NL, Barber KW, Aerni HR, Ma NJ, Haimovich AD, Rogulina S, 
            Isaacs FJ, Rinehart J
  TITLE     A flexible codon in genomically recoded Escherichia coli permits 
            programmable protein phosphorylation.
  JOURNAL   Nat Commun. 2015 Sep 9;6:8130. doi: 10.1038/ncomms9130.
  PUBMED    26350500
REFERENCE   2  (bases 1 to 4348)
  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: "Nat 
            Commun."; date: "2015-09-9"; volume: "6:8130. doi"; pages: " 
            10.1038/ncomms9130"
COMMENT     Sequence Label: GFP S2TAG/E17TAG
FEATURES             Location/Qualifiers
     source          1..4348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    38..54
                     /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)."
     protein_bind    61..77
                     /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        join(86..104,105..110,111..127,128..133,134..159)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    86..104
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    111..129
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             169..180
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             187..900
                     /codon_start=1
                     /product="GFP variant optimized for excitation by UV light"
                     /label=GFPuv
                     /translation="M*KGEELFTGVVPILV*LDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     840..860
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             907..924
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1000..1018)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1014..1061
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1158..1613
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1245..1264)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1455..1476
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1639..1743
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1744..1812,1813..2604)
                     /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     complement(1962..1981)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2775..3363
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3264..3283
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3517..3534
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(3714..4337)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     4245..4264
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 acgtcggtca gtgcgtcctg ctgatgtgct cagtatcttg ttatccgctc acaatgtcaa
       61 ttgttatccg ctcacaattc tcgagtccct atcagtgata gagattgaca tccctatcag
      121 tgatagagat actgagcaca tcagcaggac gcactgaccg aattcattaa agaggagaaa
      181 ggtaccatgt agaaaggaga agaacttttc actggagttg tcccaattct tgtttagtta
      241 gatggtgatg ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca
      301 tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca
      361 acacttgtca ctactttctc ttatggtgtt caatgctttt cccgttatcc ggatcacatg
      421 aaacggcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga acgcactata
      481 tctttcaaag atgacgggaa ctacaagacg cgtgctgaag tcaagtttga aggtgatacc
      541 cttgttaatc gtatcgagtt aaaaggtatt gattttaaag aagatggaaa cattctcgga
      601 cacaaactcg aatacaacta taactcacac aatgtataca tcacggcaga caaacaaaag
      661 aatggaatca aagctaactt caaaattcgc cacaacattg aagatggatc cgttcaacta
      721 gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac
      781 cattacctgt cgacacaatc tgccctttcg aaagatccca acgaaaagcg tgaccacatg
      841 gtccttcttg agtttgtaac tgctgctggg attacacatg gcatggatga gctctacaaa
      901 ctcgagcacc accaccacca ccactgaagg gcgagctcaa ttcaaagctt gatccggctg
      961 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1021 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1081 ccggatctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     1141 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1201 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1261 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1321 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1381 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1441 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1501 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1561 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     1621 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1681 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     1741 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     1801 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     1861 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     1921 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     1981 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2041 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2101 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     2161 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2221 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     2281 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     2341 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     2401 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     2461 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     2521 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     2581 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     2641 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     2701 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2761 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2821 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2881 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     2941 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3001 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3061 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3121 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3181 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3241 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3301 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3361 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3421 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3481 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcc
     3541 aaagggccca aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt
     3601 ccctactctc gcatggggag accccacact accatcggcg ctacggcgtt tcacttctga
     3661 gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcatct agattaagac
     3721 ccactttcac atttaagttg tttttctaat ccgcatatga tcaattcaag gccgaataag
     3781 aaggctggct ctgcaccttg gtgatcaaat aattcgatag cttgtcgtaa taatggcggc
     3841 atactatcag tagtaggtgt ttccctttct tctttagcga cttgatgctc ttgatcttcc
     3901 aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg catataatgc attctctagt
     3961 gaaaaacctt gttggcataa aaaggctaat tgattttcga gagtttcata ctgtttttct
     4021 gtaggccgtg tacctaaatg tacttttgct ccatcgcgat gacttagtaa agcacatcta
     4081 aaacttttag cgttattacg taaaaaatct tgccagcttt ccccttctaa agggcaaaag
     4141 tgagtatggt gcctatctaa catctcaatg gctaaggcgt cgagcaaagc ccgcttattt
     4201 tttacatgcc aatacaatgt aggctgctct acacctagct tctgggcgag tttacgggtt
     4261 gttaaacctt cgattccgac ctcattaagc agctctaatg cgctgttaat cactttactt
     4321 ttatctaatc tggacatgtc gaatatag
//
