LOCUS       sequence-355812-        4877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GPI-anchored RpHLuorin2 for plasma membrane expression 
            (extracellular).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4877)
  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 4877)
  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: GPI-RpHLuorin2
FEATURES             Location/Qualifiers
     source          1..4877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        92..395
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        396..599
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     550..570
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      626..635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1052..1071)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    1696..1817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1733..1752)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1787..1806
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1824..2279)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1961..1982)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2173..2192
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2306..2410
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2412..2769
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2412..2603
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2435..2455)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2620..2755
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2804..3598
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2804..3598
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2858..2877)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3468..3487
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3786..3805)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3830..3877
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4206..4794
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4695..4714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt
       61 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      121 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      181 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      241 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      301 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      361 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      421 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      481 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      541 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
      601 gtttagtgaa ccgtcagatc cgctagccac catggagctc ttttggagta tagtctttac
      661 tgtcctcctg agtttctcct gccgggggtc agactgggaa tctctgcagt cgacggtacc
      721 gcgggcccgg gatccaccgg tcgccaccat gagcaagggc gaggagctgt tcaccggcgt
      781 ggtgcccatc ctggtggagc tggacggcga cgtgaacggc cacaagttca gcgtgagcgg
      841 cgagggcgag ggcgacgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      901 caagctgccc gtgccctggc ccaccctggt gaccaccctg agctacggcg tgcagtgctt
      961 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagagcgcca tgcccgaggg
     1021 ctacgtgcag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     1081 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1141 ggaggacggc aacatcctgg gccacaagct ggagtacaac tacaacgagc acctggtcta
     1201 catcatggcc gacaagcaga agaacggcac caaggccatc ttccaggtgc accacaacat
     1261 cgaggacggc ggcgtgcagc tggccgacca ctaccagcag aacaccccca tcggcgacgg
     1321 ccccgtgctg ctgcccgaca accactacct gcacacccag agcgccctga gcaaggaccc
     1381 caacgagaag cgcgaccaca tggtgctgct ggagttcgtg accgccgccg gcatcaccca
     1441 cggcatggac gagctgtaca agggccggcc actggaaaat ggcggaacct ctctgtctga
     1501 aaaaactgtg ctgctgctgg tgactccctt tctggccgct gcttggtccc tccacccatg
     1561 aggcctcgag ggccaattaa ttaactctag atcataatca gccataccac atttgtagag
     1621 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     1681 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     1741 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     1801 ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     1861 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1921 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1981 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     2041 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     2101 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     2161 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     2221 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     2281 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2341 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2401 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     2461 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2521 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2581 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2641 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2701 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2761 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     2821 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     2881 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     2941 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     3001 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3061 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3121 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3181 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3241 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3301 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     3361 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3421 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3481 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3541 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     3601 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     3661 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     3721 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     3781 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     3841 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     3901 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     3961 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     4021 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     4081 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4141 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4201 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4261 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4321 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     4381 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4441 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4501 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4561 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4621 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4681 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4741 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4801 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4861 cctgcgttat cccctga
//
