LOCUS       sequence-445173-        4732 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter plasmid for genetic code expansion which 
            consists of a modified GFP with a Amber stop codon on position Y39.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4732)
  AUTHORS   Milles S, Tyagi S, Banterle N, Koehler C, VanDelinder V, Plass T, 
            Neal AP, Lemke EA
  TITLE     Click strategies for single-molecule protein fluorescence.
  JOURNAL   J Am Chem Soc. 2012 Mar 21;134(11):5187-95. doi: 10.1021/ja210587q. 
            Epub 2012 Mar 5.
  PUBMED    22356317
REFERENCE   2  (bases 1 to 4732)
  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/ja210587q";
            journalName: "J Am Chem Soc"; date: "2012-03-21- 21"; volume: "134";
            issue: "11"; pages: "5187-95"
COMMENT     Sequence Label: pBAD_FLAG-GFP(Y39TAG)-6His
FEATURES             Location/Qualifiers
     source          1..4732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     265..284
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     524..545
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(588..611)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(648..932)
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     complement(706..725)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             959..1837
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     primer_bind     complement(1993..2015)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2101..2241
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2156..2175
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2256..2273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2427..3015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2507..2526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3186..3977,3978..4046))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3809..3828
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4047..4138)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(4157..4184)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4276..4362)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4515..4532
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(4588..4605)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(4606..4732,1..584,585..587))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT*GKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4648..4669)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc
       61 acgctgccgt cctcgatgtt gtggcggatc ttgaagttgg ccttgatgcc gttcttctgc
      121 ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg
      181 atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg
      241 ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg
      301 cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg
      361 tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag ggtgggccag
      421 ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc ctaggtggca
      481 tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg
      541 accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcacttt atcatcatca
      601 tctttgtaat ccatggttaa ttcctcctgt tagcccaaaa aacgggtatg gagaaacagt
      661 agagagttgc gataaaaagc gtcaggtagg atccgctaat cttatggata aaaatgctat
      721 ggcatagcaa agtgtgacgc cgtgcaaata atcaatgtgg acttttctgc cgtgattata
      781 gacacttttg ttacgcgttt ttgtcatggc tttggtcccg ctttgttaca gaatgctttt
      841 aataagcggg gttaccggtt tggttagcga gaagagccag taaaagacgc agtgacggca
      901 atgtctgatg caatatggac aattggtttc ttctctgaat ggcgggagta tgaaaagtat
      961 ggctgaagcg caaaatgatc ccctgctgcc gggatactcg tttaatgccc atctggtggc
     1021 gggtttaacg ccgattgagg ccaacggtta tctcgatttt tttatcgacc gaccgctggg
     1081 aatgaaaggt tatattctca atctcaccat tcgcggtcag ggggtggtga aaaatcaggg
     1141 acgagaattt gtttgccgac cgggtgatat tttgctgttc ccgccaggag agattcatca
     1201 ctacggtcgt catccggagg ctcgcgaatg gtatcaccag tgggtttact ttcgtccgcg
     1261 cgcctactgg catgaatggc ttaactggcc gtcaatattt gccaatacgg ggttctttcg
     1321 cccggatgaa gcgcaccagc cgcatttcag cgacctgttt gggcaaatca ttaacgccgg
     1381 gcaaggggaa gggcgctatt cggagctgct ggcgataaat ctgcttgagc aattgttact
     1441 gcggcgcatg gaagcgatta acgagtcgct ccatccaccg atggataatc gggtacgcga
     1501 ggcttgtcag tacatcagcg atcacctggc agacagcaat tttgatatcg ccagcgtcgc
     1561 acagcatgtt tgcttgtcgc cgtcgcgtct gtcacatctt ttccgccagc agttagggat
     1621 tagcgtctta agctggcgcg aggaccaacg tatcagccag gcgaagctgc ttttgagcac
     1681 cacccggatg cctatcgcca ccgtcggtcg caatgttggt tttgacgatc aactctattt
     1741 ctcgcgggta tttaaaaaat gcaccggggc cagcccgagc gagttccgtg ccggttgtga
     1801 agaaaaagtg aatgatgtag ccgtcaagtt gtcataattg gtaacgaatc agacaattga
     1861 cggcttgacg gagtagcata gggtttgcag aatccctgct tcgtccattt gacaggcaca
     1921 ttatgcatgc cgcttcgcct tcgcgcgcga attgatctgc tgcctcgcgc gtttcggtga
     1981 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     2041 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     2101 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     2161 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     2221 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2281 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2341 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2401 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2461 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2521 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2581 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2641 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2701 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2761 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2821 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     2881 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2941 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3001 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3061 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3121 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3181 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3241 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3301 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3361 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3421 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3481 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3541 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3601 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3661 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3721 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3781 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa
     3841 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3901 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3961 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4021 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4081 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaaag
     4141 agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttaa tttgatgcct
     4201 ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttcg caacgttcaa
     4261 atccgctccc ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa
     4321 acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctgg cagttcccta
     4381 ctctcgcatg gggagacccc acactaccat cggcgctacg gcgtttcact tctgagttcg
     4441 gcatggggtc aggtgggacc accgcgctac tgccgccagg caaattctgt tttatcagac
     4501 cgcttctgcg ttctgattta atctgtatca ggctgaaaat cttctctcat ccgccaaaac
     4561 agccaagctc tcgagtgcgg ccgctcagtg atggtgatgg tgatgcttgt acagctcgtc
     4621 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt
     4681 ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt cg
//
