LOCUS       Exported                5875 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression of EYFP fused to Epidermal Growth Factor with a
            GST tag for purification.
ACCESSION   .
VERSION     .
KEYWORDS    SRpHi4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5875)
  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 5875)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5875
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..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             769..1485
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(814..835)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1075..1094)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1422..1443
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1743..1765)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1910..1928)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1996..2100
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2101..2961
                     /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(2319..2338)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3132..3720
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3621..3640
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3874..3891
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3964..4041
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4042..5124
                     /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(4061..4080)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        5173..5203
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5211..5227
                     /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     5235..5251
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5235..5251
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5247..5774
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5267..5284)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5267..5283)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5276..5298)
                     /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 tgggatccat ggtgagcaag
      781 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
      841 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
      901 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
      961 ttcggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1021 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1081 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1141 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1201 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1261 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1321 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     1381 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1441 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggcgg tagtggtgga
     1501 tctggcggtg aattcaatag tgactctgaa tgtcccctgt cccacgatgg gtactgcctc
     1561 catgatggtg tgtgcatgta tattgaagca ttggacaagt atgcatgcaa ctgtgttgtt
     1621 ggctacatcg gggagcgatg tcagtaccga gacctgaagt ggtgggaact gcgctaactc
     1681 gagcggccgc atcgtgactg actgacgatc tgcctcgcgc gtttcggtga tgacggtgaa
     1741 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     1801 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     1861 acccagtcac gtagcgatag cggagtgtat aattcttgaa gacgaaaggg cctcgtgata
     1921 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     1981 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2041 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2101 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     2161 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     2221 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2281 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     2341 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     2401 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     2461 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     2521 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     2581 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2641 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2701 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2761 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     2821 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     2881 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2941 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     3001 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     3061 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3121 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3181 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3241 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3301 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3361 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3421 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3481 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3541 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3601 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3661 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3721 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3781 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3841 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3901 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataaat
     3961 tccgacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     4021 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     4081 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     4141 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     4201 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     4261 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     4321 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     4381 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     4441 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     4501 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     4561 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     4621 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     4681 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     4741 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     4801 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     4861 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     4921 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     4981 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccacgct agcgcccaat
     5041 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     5101 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta
     5161 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     5221 ataacaattt cacacaggaa acagctatga ccatgattac ggattcactg gccgtcgttt
     5281 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     5341 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     5401 tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg
     5461 aaagctggct ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc
     5521 agatgcacgg ttacgatgcg cccatctaca ccaacgtgac ctatcccatt acggtcaatc
     5581 cgccgtttgt tcccacggag aatccgacgg gttgttactc gctcacattt aatgttgatg
     5641 aaagctggct acaggaaggc cagacgcgaa ttatttttga tggcgttgga attagcttat
     5701 cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc
     5761 tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat
     5821 aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctg
//
