LOCUS       sequence-243818-        5438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5438)
  AUTHORS   Koivusalo M, Welch C, Hayashi H, Scott CC, Kim M, Alexander T, 
            Touret N, Hahn KM, Grinstein S
  TITLE     Amiloride inhibits macropinocytosis by lowering submembranous pH and
            preventing Rac1 and Cdc42 signaling.
  JOURNAL   J Cell Biol. 2010 Feb 22;188(4):547-63. Epub 2010 Feb 15.
  PUBMED    20156964
REFERENCE   2  (bases 1 to 5438)
  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: "J Cell 
            Biol."; date: "2010-02-22"; volume: "188(4)"; pages: "547-63. Epub 
            2010 Feb 15"
COMMENT     Sequence Label: Lyn-tailed mCherry-SEpHluorin
FEATURES             Location/Qualifiers
     source          1..5438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        124..427
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        428..631
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     582..602
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             730..1437
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(875..893)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1143..1162
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1450..2166
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1480..1508)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2103..2123
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    2289..2410
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2326..2345)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2380..2399
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2417..2872)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2554..2575)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2766..2785
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2899..3003
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3005..3362
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3005..3196
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3028..3048)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3213..3348
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3397..4191
                     /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             3397..4191
                     /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(3451..3470)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4061..4080
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4379..4398)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4423..4470
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4799..5387
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5288..5307
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg
       61 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      121 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      181 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      241 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      301 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      361 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      421 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      481 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      541 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      601 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg
      661 ctaccggact cagatctcga gagaaatatg ggatgtatta aatcaaaaag gaaagacggg
      721 agaattccca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
      781 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
      841 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
      901 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
      961 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     1021 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     1081 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     1141 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     1201 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     1261 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     1321 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     1381 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaaggga
     1441 tccgccacca tgagtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa
     1501 ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca
     1561 acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc tgttccttgg
     1621 ccaacacttg tcactacttt aacttatggt gttcaatgct tttcaagata cccagatcat
     1681 atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact
     1741 atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat
     1801 acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt
     1861 ggacacaaat tggaatacaa ctataacgat caccaggtgt acatcatggc agacaaacaa
     1921 aagaatggaa tcaaagctaa cttcaaaatt agacacaaca ttgaagatgg aggcgttcaa
     1981 ctagcagacc attatcaaca aaatactcca attggcgatg ggcccgtcct tttaccagac
     2041 aaccattacc tgtttacaac ttctactctt tcgaaagatc ccaacgaaaa gagagaccac
     2101 atggtccttc ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac
     2161 aaataaagcg gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac
     2221 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     2281 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     2341 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     2401 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     2461 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     2521 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     2581 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     2641 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     2701 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2761 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     2821 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     2881 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2941 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3001 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3061 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3121 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3181 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3241 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3301 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3361 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3421 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3481 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3541 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     3601 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3661 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3721 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3781 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3841 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3901 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     3961 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4021 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4081 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4141 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4201 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4261 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4321 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     4381 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     4441 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     4501 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     4561 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     4621 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     4681 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4741 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4801 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4861 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4921 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4981 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5041 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5101 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5161 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5221 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5281 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5341 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5401 cggccttttt acggttcctg gccttttgct ggcctttt
//
