LOCUS       sequence-198944-        5727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of EGFP fused to the cell penetrating peptide 
            TAT with a GST tag for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5727)
  AUTHORS   Richardson DS, Gregor C, Winter FR, Urban NT, Sahl SJ, Willig KI, 
            Hell SW
  TITLE     SRpHi ratiometric pH biosensors for super-resolution microscopy.
  JOURNAL   Nat Commun. 2017 Sep 18;8(1):577. doi: 10.1038/s41467-017-00606-4.
  PUBMED    28924139
REFERENCE   2  (bases 1 to 5727)
  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: "2017-09-18"; volume: "8(1):577. doi"; pages: " 
            10.1038/s41467-017-00606-4"
COMMENT     Sequence Label: SRpHi2
FEATURES             Location/Qualifiers
     source          1..5727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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)."
     CDS             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..759
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(808..810,811..1524)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(808..810,811..1521)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(850..871)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1111..1130)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1458..1479
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1595..1617)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1762..1780)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1848..1952
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1953..2021,2022..2813)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2171..2190)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2984..3572
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3473..3492
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3726..3743
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3816..3893
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3894..4976
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3913..3932)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5025..5030,5031..5048,5049..5055)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5063..5079
                     /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)."
     primer_bind     5087..5103
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5087..5103
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5099..5626
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5119..5136)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5119..5135)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5128..5150)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctgga agttctgttc caggggcccc tgggatccta tggccgaaag
      781 aagcggcgtc aacgacgtcg agaattcgtg agcaagggcg aggagctgtt caccggggtg
      841 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
      901 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
      961 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1021 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1081 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1141 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1201 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1261 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     1321 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1381 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     1441 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1501 ggcatggacg agctgtacaa gtaactcgac tcgagcggcc gcatcgtgac tgactgacga
     1561 tctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     1621 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     1681 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     1741 ataattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1801 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1861 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1921 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1981 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2041 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2101 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2161 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2221 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2281 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2341 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2401 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2461 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     2521 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2581 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2641 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2701 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2761 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2821 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2881 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2941 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3001 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3061 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3121 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3181 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3241 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3301 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3361 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3421 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3481 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3541 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3601 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3661 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3721 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3781 cgcatctgtg cggtatttca caccgcataa attccgacac catcgaatgg cgcaaaacct
     3841 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     3901 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     3961 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     4021 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     4081 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     4141 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     4201 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     4261 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     4321 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     4381 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     4441 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     4501 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     4561 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     4621 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     4681 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     4741 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     4801 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     4861 tggtgaaaag aaaaaccacg ctagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     4921 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     4981 aacgcaatta atgtaagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     5041 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     5101 gaccatgatt acggattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     5161 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     5221 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt
     5281 tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga
     5341 ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac ggttacgatg cgcccatcta
     5401 caccaacgtg acctatccca ttacggtcaa tccgccgttt gttcccacgg agaatccgac
     5461 gggttgttac tcgctcacat ttaatgttga tgaaagctgg ctacaggaag gccagacgcg
     5521 aattattttt gatggcgttg gaattagctt atcgactgca cggtgcacca atgcttctgg
     5581 cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt
     5641 cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg
     5701 ttctggcaaa tattctgaaa tgagctg
//
