LOCUS       sequence-468197-        6064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of recombinant His-tagged E1GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6064)
  AUTHORS   De Lorenzi V, Ghignoli S, Bernardi M, Matteoli G, Ferri G, Storti B,
            Bizzarri R, Cardarelli F
  TITLE     Measuring pH in insulin secretory granules by phasor-based 
            fluorescence lifetime imaging of a genetically encoded sensor.
  JOURNAL   Commun Biol. 2025 Feb 25;8(1):304. doi: 10.1038/s42003-025-07758-w.
  PUBMED    40000885
REFERENCE   2  (bases 1 to 6064)
  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: "Commun 
            Biol."; date: "2025-02-25"; volume: "8(1):304. doi"; pages: " 
            10.1038/s42003-025-07758-w"
COMMENT     Sequence Label: pET-His-E1GFP
FEATURES             Location/Qualifiers
     source          1..6064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(219..932,933..935,936..938))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(264..285)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     613..632
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     872..893
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(942..959)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(969..986)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1005..1027
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1042..1066
                     /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     complement(1066..1085)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1067..1085)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1169..1188)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1394..1471
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1472..2554
                     /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(1491..1510)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3363..3554
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3548..3570)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3656..3795
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3711..3730
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3810..3827)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3981..4569)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4061..4080)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4680..4699)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4691..5506
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4691..5506
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4764..4783)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5598..6053)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5735..5756)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5947..5966
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccctgaaaa taaagattct acttgtacag ctcgtccatg
      241 ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg
      301 ttggggtctt tgctcagggc ggactggtag ctcaggtagt ggttgtcggg cagcagcacg
      361 gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc
      421 tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt gtcggccatg
      481 atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat gttgccgtcc
      541 tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc
      601 acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg ctcctggacg
      661 tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg
      721 ctgaagcact gcacgccgta gctcagggtg gtcacgaggg tgggccaggg cacgggcagc
      781 ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc gccctcgccc
      841 tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac caggatgggc
      901 accaccccgg tgaacagctc ctcgcccttg ctcaccatat ggctgccgcg cggcaccagg
      961 ccgctgctgt gatgatgatg atgatggctg ctgcccatgg tatatctcct tcttaaagtt
     1021 aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg
     1081 tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc
     1141 accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat
     1201 cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc
     1261 gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg
     1321 ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag
     1381 cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg
     1441 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     1501 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     1561 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     1621 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     1681 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     1741 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     1801 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     1861 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     1921 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     1981 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     2041 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     2101 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     2161 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     2221 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
     2281 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     2341 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     2401 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     2461 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2521 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     2581 tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct
     2641 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     2701 tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc
     2761 gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg
     2821 ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg
     2881 ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag
     2941 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     3001 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     3061 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
     3121 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
     3181 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
     3241 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
     3301 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     3361 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     3421 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     3481 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     3541 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     3601 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     3661 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     3721 gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga
     3781 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3841 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3901 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3961 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4021 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4081 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4141 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4201 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4261 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4321 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4381 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     4441 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4501 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4561 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4621 tcacgttaag ggattttggt catgaacaat aaaactgtct gcttacataa acagtaatac
     4681 aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc
     4741 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     4801 tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     4861 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     4921 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     4981 caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     5041 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     5101 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     5161 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     5221 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     5281 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     5341 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     5401 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     5461 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat taattcatga
     5521 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5581 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     5641 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5701 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5761 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     5821 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     5881 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5941 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     6001 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     6061 gcca
//
