LOCUS       sequence-54243-e        6839 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 6839)
  AUTHORS   Toth DJ, Toth JT, Gulyas G, Balla A, Balla T, Hunyady L, Varnai P
  TITLE     Acute depletion of plasma membrane phosphatidylinositol 
            4,5-bisphosphate impairs specific steps in endocytosis of the 
            G-protein-coupled receptor.
  JOURNAL   J Cell Sci. 2012 May 1;125(Pt 9):2185-97. Epub 2012 Feb 22.
  PUBMED    22357943
REFERENCE   2  (bases 1 to 6839)
  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 
            Sci."; date: "2012-05-1"; volume: "125(Pt 9)"; pages: "2185-97. Epub
            2012 Feb 22"
COMMENT     Sequence Label: PM-FRB-Cerulean-T2A-FKBP-5-ptase
FEATURES             Location/Qualifiers
     source          1..6839
                     /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"
     CDS             complement(670..687)
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             702..980
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             987..1013
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1020..1029
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1026..1028,1029..1031,1032..1742)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1071..1092)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1332..1351)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1679..1700
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1758..1811
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      1818..1827
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1824..1826,1827..2147)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     polyA_signal    3627..3748
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3664..3683)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3718..3737
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3755..4210)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3892..3913)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4104..4123
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4237..4341
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4343..4700
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4343..4534
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4366..4386)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4551..4686
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4735..5529
                     /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             4735..5529
                     /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(4789..4808)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5399..5418
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5717..5736)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5761..5808
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6137..6725
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6626..6645
                     /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 gctagcacca
      601 tgggatgtat aaaatcaaaa gggaaagaca gcgcgggagc agatcccacc agatctgcga
      661 attcaggtgc tggtgctggt gctggtgcta tcctatctag aatcctctgg catgagatgt
      721 ggcatgaagg cctggaagag gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca
      781 tgtttgaggt gctggagccc ttgcatgcta tgatggaacg gggcccccag actctgaagg
      841 aaacatcctt taatcaggcc tatggtcgag atttaatgga ggcccaagag tggtgcagga
      901 agtacatgaa atcagggaat gtcaaggacc tcacccaagc ctgggacctc tattatcatg
      961 tgttccgacg aatctcaaag actagttatc cgtacgacgt accagactac gcaccggtcg
     1021 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1081 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1141 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1201 ccaccctcgt gaccaccctg acctggggcg tgcagtgctt cgcccgctac cccgaccaca
     1261 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1321 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1381 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1441 ggcacaagct ggagtacaac gccatcagcg acaacgtcta tatcaccgcc gacaagcaga
     1501 agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1561 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1621 accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     1681 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1741 aggtcgactc cgggagtgag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga
     1801 atcccggccc accggtcgcc accatgggag tgcaggtgga aaccatctcc ccaggagacg
     1861 ggcgcacctt ccccaagcgc ggccagacct gcgtggtgca ctacaccggg atgcttgaag
     1921 atggaaagaa atttgattcc tcccgggaca gaaacaagcc ctttaagttt atgctaggca
     1981 agcaggaggt gatccgaggc tgggaagaag gggttgccca gatgagtgtg ggtcagagag
     2041 ccaaactgac tatatctcca gattatgcct atggtgccac tgggcaccca ggcatcatcc
     2101 caccacatgc cactctcgtc ttcgatgtgg agcttctaaa actggaagct cgaggcgcag
     2161 cagcgggagc aggaggagca ggtcgatcgg atcttgcaga ctacaagctc cgcgcgcagc
     2221 cgctcctggt gcgggcccac agcagcctgg gccccggccg gccgcggagc cccctggcct
     2281 gcgacgactg ttcccttcgc tcggccaaat cctccttcag cctcctggcg cccatccgca
     2341 gcaaggacgt ccgcagcagg agttacctgg agggcagcct cctggccagc ggggccctgt
     2401 tgggggcgga tgagctggcc cgctacttcc cagaccggaa cgtggcactc ttcgtggcca
     2461 cctggaacat gcagggccag aaggagctcc cgcccagcct ggacgagttc ctgctcccag
     2521 ccgaggccga ctatgcccag gacctgtatg tcatcggggt ccaggagggc tgttctgaca
     2581 ggcgggagtg ggagactcgt ctgcaggaga cgctgggccc gcactatgtg ctgctgtcct
     2641 cggcggccca cggcgtgctc tacatgtcgc tcttcatccg cagggacctc atctggttct
     2701 gctcagaggt ggagtgctcc acggtgacca cacgcatcgt gtctcagatc aagaccaagg
     2761 gggccttggg catcagcttc accttttttg gcacttcctt cctcttcatc acgtcccact
     2821 tcacctcagg tgacgggaag gtggcggagc ggctgctgga ctacaccagg actgtacaag
     2881 ccctggtcct gcccagaaat gtgcccgaca ccaaccccta tcgctccagc gcagcggacg
     2941 tcaccacccg cttcgatgag gtgttctggt ttggagactt caacttccgc ctgagtggcg
     3001 ggcgcacagt cgtggacgcc ctcctgtgcc agggcctggt ggtggacgtg ccggcgctgc
     3061 tgcagcacga ccagctcatc cgggagatgc ggaaagggtc catcttcaag ggcttccagg
     3121 agccggacat ccacttcctc ccatcataca agtttgacat cgggaaggac acgtacgaca
     3181 gcacctccaa gcagaggacg ccctcataca cggaccgcgt cttgtacaga agccgccaca
     3241 agggtgacat ctgtcctgtg agctactctt cctgccccgg gatcaagacg tccgaccacc
     3301 gccctgtgta tggcctcttc cgggtgaaag tgaggccggg gcgagacaac attccgttgg
     3361 cagctggcaa atttgataga gaactgtact tactaggaat taaaagacgg atttcgaagg
     3421 agattcagag gcagcaagca ctacagagtc agaactccag caccatcgcc tccgtttctt
     3481 gaggtaccgc gggcccggga tccaccggat ctagataact gatcataatc agccatacca
     3541 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     3601 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     3661 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3721 gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta
     3781 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     3841 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     3901 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     3961 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4021 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4081 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4141 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4201 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4261 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4321 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     4381 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4441 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4501 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4561 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4621 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4681 gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4741 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4801 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4861 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     4921 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4981 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     5041 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     5101 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     5161 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5221 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     5281 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5341 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5401 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5461 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5521 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5581 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5641 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5701 ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     5761 cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg
     5821 ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg
     5881 cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg
     5941 cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga
     6001 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6061 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6121 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6181 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6241 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     6301 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6361 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6421 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6481 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6541 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6601 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6661 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6721 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6781 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
