LOCUS       sequence-355807-        6003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for bacterial expression of recombinant ratiometric pHluorin2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6003)
  AUTHORS   Linders PTA, Ioannidis M, Ter Beest M, van den Bogaart G
  TITLE     Fluorescence Lifetime Imaging of pH along the Secretory Pathway.
  JOURNAL   ACS Chem Biol. 2022 Jan 21;17(1):240-251. doi: 
            10.1021/acschembio.1c00907. Epub 2022 Jan 10.
  PUBMED    35000377
REFERENCE   2  (bases 1 to 6003)
  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/acschembio.1c00907"; journalName: "ACS Chem Biol"; date: 
            "2022-01-21- 21"; volume: "17"; issue: "1"; pages: "240-251"
COMMENT     Sequence Label: RpHLuorin2
FEATURES             Location/Qualifiers
     source          1..6003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     845..864
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(884..961)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1167..1186
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1270..1289
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1270..1288
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1289..1313
                     /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)."
     RBS             1328..1350
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1369..1386
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1396..1413
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2137..2154
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2207..2225)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2221..2268
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2305..2760
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2392..2411)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2602..2623
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2852..3667)
                     /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             complement(2852..3667)
                     /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     3575..3594
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3659..3678
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3789..4377
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4278..4297
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4531..4548
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4563..4702
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4628..4647)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4788..4810
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4804..4995)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5804..6003,1..883))
                     /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"
ORIGIN
        1 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
       61 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      121 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      181 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      241 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      301 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      361 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      421 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      481 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      541 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      601 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      661 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      721 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      781 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      841 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
      901 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
      961 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1021 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1081 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1141 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1201 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     1261 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1321 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
     1381 catcacagca gcggcctggt gccgcgcggc agccatatgt ctaaaggtga agaactgttc
     1441 accggtgttg ttccgatcct ggttgaactg gacggtgacg ttaacggtca caaattctct
     1501 gtttctggtg aaggtgaagg tgacgctacc tacggtaaac tgaccctgaa attcatctgc
     1561 accaccggta aactgccggt tccgtggccg accctggtta ccaccctgtc ttacggtgtt
     1621 cagtgcttct ctcgttaccc ggaccacatg aaacagcacg acttcttcaa atctgctatg
     1681 ccggaaggtt acgttcagga acgtaccatc ttcttcaaag acgacggtaa ctacaaaacc
     1741 cgtgctgaag ttaaattcga aggtgacacc ctggttaacc gtatcgaact gaaaggtatc
     1801 gacttcaaag aagacggtaa catcctgggt cacaaactgg aatacaacta caacgaacac
     1861 ctggtttaca tcatggctga caaacagaaa aacggtacca aagctatctt ccaggttcac
     1921 cacaacatcg aagacggtgg tgttcagctg gctgaccact accagcagaa caccccgatc
     1981 ggtgacggtc cggttctgct gccggacaac cactacctgc acacccagtc tgctctgtct
     2041 aaagacccga acgaaaaacg tgaccacatg gttctgctgg aattcgttac cgctgctggt
     2101 atcacccacg gtatggacga actgtacaaa ctcgagcacc accaccacca ccactgagat
     2161 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2221 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2281 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2341 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2401 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2461 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2521 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2581 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2641 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2701 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2761 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2821 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2881 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2941 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3001 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3061 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     3121 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3181 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3241 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3301 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3361 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3421 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3481 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3541 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3601 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3661 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3721 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3781 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3841 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3901 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3961 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4021 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4081 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4141 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4201 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4261 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4321 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4381 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4441 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4501 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4561 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4621 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4681 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4741 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4801 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4861 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4921 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4981 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     5041 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     5101 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     5161 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     5221 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5281 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5341 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     5401 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5461 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5521 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5581 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5641 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5701 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5761 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5821 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5881 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5941 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     6001 gca
//
