LOCUS       sequence-380177-        5849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human Clathrin Light Chain B tagged with SNAP-tag and 
            mEGFP in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5849)
  AUTHORS   Obashi K, Sochacki KA, Strub MP, Taraska JW
  TITLE     A conformational switch in clathrin light chain regulates lattice 
            structure and endocytosis at the plasma membrane of mammalian cells.
  JOURNAL   Nat Commun. 2023 Feb 9;14(1):732. doi: 10.1038/s41467-023-36304-7.
  PUBMED    36759616
REFERENCE   2  (bases 1 to 5849)
  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: "Nat 
            Commun."; date: "2023-02-9"; volume: "14(1):732. doi"; pages: " 
            10.1038/s41467-023-36304-7"
COMMENT     Sequence Label: SNAP-CLC-EGFP
FEATURES             Location/Qualifiers
     source          1..5849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        72..375
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        376..579
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     530..550
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      618..627
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             624..1169
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for mammalian expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(1866..1887)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2127..2146)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2286..2537
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     2474..2495
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2648..2769
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2685..2704)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2739..2758
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2776..3231)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2913..2934)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3125..3144
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3258..3362
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3364..3721
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3364..3555
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3387..3407)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3572..3707
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3756..4550
                     /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             3756..4550
                     /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(3810..3829)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4420..4439
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4738..4757)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4782..4829
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5158..5746
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5647..5666
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgccatgca ttagttatta atagtaatca attacggggt cattagttca tagcccatat
       61 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      121 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      181 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      241 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      301 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      361 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
      421 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      481 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
      541 ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc
      601 cgctagcgct accggtcgcc accatggaca aagactgcga aatgaagcgc accaccctgg
      661 atagccctct gggcaagctg gaactgtctg ggtgcgaaca gggcctgcac gagatcaagc
      721 tgctgggcaa aggaacatct gccgccgacg ccgtggaagt gcctgcccca gccgccgtgc
      781 tgggcggacc agagccactg atgcaggcca ccgcctggct caacgcctac tttcaccagc
      841 ctgaggccat cgaggagttc cctgtgccag ccctgcacca cccagtgttc cagcaggaga
      901 gctttacccg ccaggtgctg tggaaactgc tgaaagtggt gaagttcgga gaggtcatca
      961 gctaccagca gctggccgcc ctggccggca atcccgccgc caccgccgcc gtgaaaaccg
     1021 ccctgagcgg aaatcccgtg cccattctga tcccctgcca ccgggtggtg tctagctctg
     1081 gcgccgtggg gggctacgag ggcgggctcg ccgtgaaaga gtggctgctg gcccacgagg
     1141 gccacagact gggcaagcct gggctgggtg gatccggagg gtctatggct gatgactttg
     1201 gcttcttctc gtcgtcggag agtggtgccc cggaggcggc ggaggaggac ccggcggccg
     1261 ccttcctggc ccagcaggag agcgagattg caggcataga gaacgacgag ggcttcgggg
     1321 cacctgccgg cagccatgcg gcccccgcac agccgggccc cacgagtggg gctggttctg
     1381 aggacatggg gaccacagtc aatggagatg tgtttcagga ggccaacggt cctgctgatg
     1441 gctacgcagc cattgcccag gctgacaggc tgacccagga gcctgagagc atccgcaagt
     1501 ggcgagagga gcagaggaaa cggctgcaag agctggatgc tgcatctaag gtcacggaac
     1561 aggaatggcg ggagaaggcc aagaaggacc tggaggagtg gaaccagcgc cagagtgaac
     1621 aagtagagaa gaacaagatc aacaaccggg catccgagga ggctttcgtg aaggaatcca
     1681 aggaggagac cccaggcaca gagtgggaga aggtggccca gctatgtgac ttcaacccca
     1741 agagcagcaa gcagtgcaaa gatgtgtccc gcctgcgctc ggtgctcatg tccctgaagc
     1801 agacgccact gtcccgcggc ggatccggag ggtctgagga gctgttcacc ggggtggtgc
     1861 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1921 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1981 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     2041 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     2101 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     2161 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     2221 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     2281 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     2341 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     2401 tgctgctgcc cgacaaccac tacctgagca cccagtccaa gctgagcaaa gaccccaacg
     2461 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2521 tggacgagct gtacaagtaa ctgatcataa tcagccatac acatttgtag aggttttact
     2581 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2641 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2701 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2761 tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     2821 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     2881 aagaatagac cgagatcggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2941 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     3001 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     3061 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     3121 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     3181 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     3241 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3301 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3361 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3421 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3481 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3541 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3601 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3661 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3721 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3781 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3841 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3901 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     3961 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     4021 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     4081 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     4141 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     4201 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4261 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     4321 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4381 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4441 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4501 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4561 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4621 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4681 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga
     4741 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     4801 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca
     4861 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc
     4921 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc
     4981 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt
     5041 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5101 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5161 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5221 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5281 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5341 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5401 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5461 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5521 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5581 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5641 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5701 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5761 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5821 atcccctgat tctgtggata accgtatta
//
