LOCUS       sequence-235329-        4777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the proton pump rhodopsin in mammalian cells under CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4777)
  AUTHORS   Wu L, Dong A, Dong L, Wang SQ, Li Y
  TITLE     PARIS, an optogenetic method for functionally mapping gap junctions.
  JOURNAL   Elife. 2019 Jan 14;8. pii: 43366. doi: 10.7554/eLife.43366.
  PUBMED    30638447
REFERENCE   2  (bases 1 to 4777)
  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: "Elife."; 
            date: "2019-01-14"; volume: "8. pii: 43366. doi"; pages: " 
            10.7554/eLife.43366"
COMMENT     Sequence Label: pN3-pHluorinCAAX
FEATURES             Location/Qualifiers
     source          1..4777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    609..665
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      669..678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             675..1391
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(708..736)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1331..1351
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    1565..1686
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1602..1621)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1656..1675
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1693..2148)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1830..1851)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2042..2061
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2175..2279
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2281..2638
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2281..2472
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2304..2324)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2489..2624
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2673..3467
                     /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             2673..3467
                     /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(2727..2746)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3337..3356
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3655..3674)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3699..3746
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4075..4663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4564..4583
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccatcgc caccatggtg agtaaaggag aagaactttt cactggagtt gtcccaattc
      721 ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag
      781 gtgatgcaac atacggaaaa cttaccctta aatttatttg cactactgga aaactacctg
      841 ttccttggcc aacacttgtc actactttaa cttatggtgt tcaatgcttt tcaagatacc
      901 cagatcatat gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg
      961 aaagaactat atttttcaaa gatgacggga actacaagac acgtgctgaa gtcaagtttg
     1021 aaggtgatac ccttgttaat agaatcgagt taaaaggtat tgattttaaa gaagatggaa
     1081 acattcttgg acacaaattg gaatacaact ataacgatca ccaggtgtac atcatggcag
     1141 acaaacaaaa gaatggaatc aaagctaact tcaaaattag acacaacatt gaagatggag
     1201 gcgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggg cccgtccttt
     1261 taccagacaa ccattacctg tttacaactt ctactctttc gaaagatccc aacgaaaaga
     1321 gagaccacat ggtccttctt gagtttgtaa cagctgctgg gattacacat ggcatggatg
     1381 aactatacaa agatggtaaa aagaagaaaa agaagtcaaa gacaaagtgt gtaattatgt
     1441 aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     1501 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     1561 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1621 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1681 atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     1741 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     1801 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     1861 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     1921 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     1981 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     2041 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     2101 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     2161 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2221 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     2281 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2341 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2401 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2461 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2521 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     2581 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     2641 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     2701 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     2761 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     2821 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     2881 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     2941 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3001 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3061 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3121 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3181 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3241 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3301 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3361 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3421 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     3481 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     3541 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3601 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     3661 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     3721 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     3781 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     3841 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     3901 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     3961 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4021 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4081 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4141 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4201 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     4261 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4321 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4381 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4441 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4501 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4561 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4621 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4681 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4741 ccctgattct gtggataacc gtattaccgc catgcat
//
