LOCUS       sequence-54241-e        6797 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 6797)
  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 6797)
  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-mRFP-T2A-FKBP-5-ptase
FEATURES             Location/Qualifiers
     source          1..6797
                     /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"
     CDS             1029..1700
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1029..1700
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(1156..1174)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1424..1443
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1618..1641
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1716..1769
                     /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      1776..1785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1782..1784,1785..2105)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     polyA_signal    3585..3706
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3622..3641)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3676..3695
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3713..4168)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3850..3871)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4062..4081
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4195..4299
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4301..4658
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4301..4492
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4324..4344)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4509..4644
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4693..5487
                     /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             4693..5487
                     /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(4747..4766)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5357..5376
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5675..5694)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5719..5766
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6095..6683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6584..6603
                     /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 ccaccctggc ctcctccgag gacgtcatca aggagttcat gcgcttcaag gtgcgcatgg
     1081 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1141 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
     1201 acatcctgtc ccctcagttc cagtacggct ccaaggccta cgtgaagcac cccgccgaca
     1261 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1321 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     1381 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     1441 agaccatggg ctgggaggcc tccaccgagc ggatgtaccc cgaggacggc gccctgaagg
     1501 gcgagatcaa gatgaggctg aagctgaagg acggcggcca ctacgacgcc gaggtcaaga
     1561 ccacctacat ggccaagaag cccgtgcagc tgcccggcgc ctacaagacc gacatcaagc
     1621 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgcgccgagg
     1681 gccgccactc caccggcgcc gtcgactccg ggagtgaggg cagaggaagt cttctaacat
     1741 gcggtgacgt ggaggagaat cccggcccac cggtcgccac catgggagtg caggtggaaa
     1801 ccatctcccc aggagacggg cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact
     1861 acaccgggat gcttgaagat ggaaagaaat ttgattcctc ccgggacaga aacaagccct
     1921 ttaagtttat gctaggcaag caggaggtga tccgaggctg ggaagaaggg gttgcccaga
     1981 tgagtgtggg tcagagagcc aaactgacta tatctccaga ttatgcctat ggtgccactg
     2041 ggcacccagg catcatccca ccacatgcca ctctcgtctt cgatgtggag cttctaaaac
     2101 tggaagctcg aggcgcagca gcgggagcag gaggagcagg tcgatcggat cttgcagact
     2161 acaagctccg cgcgcagccg ctcctggtgc gggcccacag cagcctgggc cccggccggc
     2221 cgcggagccc cctggcctgc gacgactgtt cccttcgctc ggccaaatcc tccttcagcc
     2281 tcctggcgcc catccgcagc aaggacgtcc gcagcaggag ttacctggag ggcagcctcc
     2341 tggccagcgg ggccctgttg ggggcggatg agctggcccg ctacttccca gaccggaacg
     2401 tggcactctt cgtggccacc tggaacatgc agggccagaa ggagctcccg cccagcctgg
     2461 acgagttcct gctcccagcc gaggccgact atgcccagga cctgtatgtc atcggggtcc
     2521 aggagggctg ttctgacagg cgggagtggg agactcgtct gcaggagacg ctgggcccgc
     2581 actatgtgct gctgtcctcg gcggcccacg gcgtgctcta catgtcgctc ttcatccgca
     2641 gggacctcat ctggttctgc tcagaggtgg agtgctccac ggtgaccaca cgcatcgtgt
     2701 ctcagatcaa gaccaagggg gccttgggca tcagcttcac cttttttggc acttccttcc
     2761 tcttcatcac gtcccacttc acctcaggtg acgggaaggt ggcggagcgg ctgctggact
     2821 acaccaggac tgtacaagcc ctggtcctgc ccagaaatgt gcccgacacc aacccctatc
     2881 gctccagcgc agcggacgtc accacccgct tcgatgaggt gttctggttt ggagacttca
     2941 acttccgcct gagtggcggg cgcacagtcg tggacgccct cctgtgccag ggcctggtgg
     3001 tggacgtgcc ggcgctgctg cagcacgacc agctcatccg ggagatgcgg aaagggtcca
     3061 tcttcaaggg cttccaggag ccggacatcc acttcctccc atcatacaag tttgacatcg
     3121 ggaaggacac gtacgacagc acctccaagc agaggacgcc ctcatacacg gaccgcgtct
     3181 tgtacagaag ccgccacaag ggtgacatct gtcctgtgag ctactcttcc tgccccggga
     3241 tcaagacgtc cgaccaccgc cctgtgtatg gcctcttccg ggtgaaagtg aggccggggc
     3301 gagacaacat tccgttggca gctggcaaat ttgatagaga actgtactta ctaggaatta
     3361 aaagacggat ttcgaaggag attcagaggc agcaagcact acagagtcag aactccagca
     3421 ccatcgcctc cgtttcttga ggtaccgcgg gcccgggatc caccggatct agataactga
     3481 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     3541 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     3601 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3661 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta
     3721 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     3781 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     3841 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     3901 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     3961 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     4021 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     4081 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     4141 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     4201 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4261 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     4321 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     4381 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     4441 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     4501 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     4561 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     4621 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     4681 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4741 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4801 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4861 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4921 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4981 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     5041 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     5101 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     5161 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     5221 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     5281 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     5341 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5401 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     5461 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     5521 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5581 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5641 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     5701 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     5761 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     5821 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     5881 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     5941 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6001 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6061 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6121 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6181 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6241 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6301 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6361 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6421 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6481 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6541 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6601 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6661 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6721 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6781 gtattaccgc catgcat
//
