LOCUS       sequence-198945-        5722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of EYFP 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 5722)
  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 5722)
  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: SRpHi1
FEATURES             Location/Qualifiers
     source          1..5722
                     /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="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     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(1590..1612)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1757..1775)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1843..1947
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1948..2016,2017..2808)
                     /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(2166..2185)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2979..3567
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3468..3487
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3721..3738
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3811..3888
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3889..4971
                     /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(3908..3927)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5020..5025,5026..5043,5044..5050)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5058..5074
                     /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     5082..5098
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5082..5098
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5094..5621
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5114..5131)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5114..5130)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5123..5145)
                     /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 accaccttcg gctacggcct gcagtgcttc
     1021 gcccgctacc 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 agctaccagt ccgccctgag caaagacccc
     1441 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1501 ggcatggacg agctgtacaa gtaactcgag cggccgcatc gtgactgact gacgatctgc
     1561 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     1621 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     1681 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat
     1741 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     1801 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1861 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1921 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1981 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2041 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2101 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2161 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     2221 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2281 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2341 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2401 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2461 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     2521 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2581 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2641 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2701 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2761 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2821 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2881 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2941 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3001 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3061 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3121 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3181 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3241 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3301 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3361 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3421 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3481 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3541 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3601 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3661 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3721 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     3781 ctgtgcggta tttcacaccg cataaattcc gacaccatcg aatggcgcaa aacctttcgc
     3841 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     3901 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     3961 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     4021 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     4081 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     4141 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     4201 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     4261 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     4321 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     4381 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     4441 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     4501 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     4561 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     4621 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     4681 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     4741 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     4801 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     4861 aaaagaaaaa ccacgctagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     4921 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     4981 aattaatgta agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     5041 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     5101 tgattacgga ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     5161 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     5221 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct
     5281 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg
     5341 atactgtcgt cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca
     5401 acgtgaccta tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt
     5461 gttactcgct cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta
     5521 tttttgatgg cgttggaatt agcttatcga ctgcacggtg caccaatgct tctggcgtca
     5581 ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt
     5641 cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg
     5701 gcaaatattc tgaaatgagc tg
//
