LOCUS       sequence-391846-        5756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Visualization of clathrin-coated pit formation in living mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5756)
  AUTHORS   Sakaushi S, Senda-Murata K, Fukada T, Oka S, Sugimoto K
  TITLE     Rhythmic cycle of clathrin-coated pit formation at the trans-golgi 
            network in human MDA-MB-435 cells.
  JOURNAL   Biosci Biotechnol Biochem. 2007 Feb;71(2):571-4. doi: 
            10.1271/bbb.60434. Epub 2007 Feb 7.
  PUBMED    17284835
REFERENCE   2  (bases 1 to 5756)
  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: "10.1271/bbb.60434";
            journalName: "Biosci Biotechnol Biochem"; date: "2007-02"; volume: 
            "71"; issue: "2"; pages: "571-4"
COMMENT     Sequence Label: pEGFP-CLCa
FEATURES             Location/Qualifiers
     source          1..5756
                     /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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2544..2665
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2581..2600)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2635..2654
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2672..3127)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2809..2830)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3021..3040
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3154..3258
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3260..3617
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3260..3451
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3283..3303)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3468..3603
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3652..4446
                     /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             3652..4446
                     /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(3706..3725)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4316..4335
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4634..4653)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4678..4725
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5054..5642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5543..5562
                     /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 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct caagctagct tcgaattcgg cacgagcggt
     1381 gggtcggttg gtttttgtct caccgttggt gtccgtgccg ttcagttgcc cgccatggct
     1441 gagctggatc cgttcggcgc ccctgccggc gcccctggcg gtcccgcgct ggggaacgga
     1501 gtggccggcg ccggcgaaga agacccggct gcggccttct tggcgcagca agagagcgag
     1561 attgcgggca tcgagaacga cgaggccttc gccatcctgg acggcggcgc ccccgggccc
     1621 cagccgcacg gcgagccgcc ggggggtccg gatgctgttg atggagtaat gaatggtgaa
     1681 tactaccagg aaagtaatgg tccaacagac agttatgcag ctatttcaca agtggatcga
     1741 ttgcagtcag agcctgaaag tatccgtaaa tggagagaag aacaaatgga acgcttggaa
     1801 gcccttgatg ccaattctcg gaagcaagaa gcagagtgga aagaaaaggc aataaaggag
     1861 ctagaagaat ggtatgcaag acaggacgag cagctacaga aaacaaaagc aaacaacagg
     1921 gcagcagaag aagcctttgt aaatgacatt gacgagtcgt ccccaggcac tgagtgggaa
     1981 cgggtggccc ggctgtgtga ctttaacccc aagtctagca agcaggccaa agatgtctcc
     2041 cgcatgcgct cagtcctcat ctccctcaag caggccccgc tggtgcactg aagagccacc
     2101 ctgtggaaac actacatctg caatatctta atcctactca gtgaagctct tcacagtcat
     2161 tggattaatt atgttgagtt cttttggacc aaaccttttt gtctttagag ttgttcattg
     2221 tttgtgattg catgtttcct tccttcaact gtgttctccc tggcattcag agaggaggga
     2281 gaggaggaag aggaagggga gggaagcttc ccaagagtag cctcaacctg tgcttctgtg
     2341 cattattctg agaataaatt tctgtttcaa actgtaaaaa aaaaaaaaaa aaactcgacg
     2401 gtaccgcggg cccgggatcc accggatcta gataactgat cataatcagc cataccacat
     2461 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     2521 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     2581 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     2641 tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa
     2701 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2761 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2821 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     2881 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2941 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3001 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3061 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3121 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3181 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3241 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     3301 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3361 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3421 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3481 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3541 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3601 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     3661 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3721 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3781 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     3841 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3901 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3961 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4021 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4081 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4141 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     4201 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4261 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4321 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4381 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4441 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4501 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4561 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     4621 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     4681 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     4741 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     4801 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     4861 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     4921 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4981 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5041 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5101 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5161 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5221 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5281 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5341 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5401 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5461 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5521 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5581 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5641 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5701 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
