LOCUS       sequence-443319-        8157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CLCN3 expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8157)
  AUTHORS   Gonzalez-Lozano MA, Schmid EW, Miguel Whelan E, Jiang Y, Paulo JA, 
            Walter JC, Harper JW
  TITLE     EndoMAP.v1 charts the structural landscape of human early endosome 
            complexes.
  JOURNAL   Nature. 2025 Jul;643(8070):252-261. doi: 10.1038/s41586-025-09059-y.
            Epub 2025 May 28.
  PUBMED    40437099
REFERENCE   2  (bases 1 to 8157)
  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: 
            "10.1038/s41586-025-09059-y"; journalName: "Nature"; date: 
            "2025-07"; volume: "643"; issue: "8070"; pages: "252-261"
COMMENT     Sequence Label: pcDNA -CLCN3-3xFlag
FEATURES             Location/Qualifiers
     source          1..8157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1291..2316)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(1291..2316)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(2365..2694)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2379..2514
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2433..2452)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2679..2699
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2708..3136)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2818..2839)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3030..3049
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3285..3396
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3385..3402
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3446..3511)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    3513..3537
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5998..6022)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(6109..6128)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6110..6128)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6173..6376)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6202..6222)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6377..6756
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6928..6947
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        7022..7126
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7127..7195,7196..7987)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7345..7364)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtagcacgtg ctattccttt gccctcggac gagtgctggg
     1321 gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct
     1381 tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca
     1441 tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg
     1501 gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg
     1561 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa
     1621 gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc
     1681 tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg
     1741 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga
     1801 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc
     1861 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa
     1921 cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atggcctccg cgaccggctg
     1981 cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
     2041 ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg
     2101 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca
     2161 gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc
     2221 ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt
     2281 ctcgacagac gtcgcggtga gttcaggctt tttcatcacg tgctgatcag atccgaaaat
     2341 ggatatacaa gctcccggga gctttttgca aaagcctagg cctccaaaaa agcctcctca
     2401 ctacttctgg aatagctcag aggcagaggc ggcctcggcc tctgcataaa taaaaaaaat
     2461 tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg
     2521 agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc
     2581 tgctggggag cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg
     2641 catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca
     2701 cagaattaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2761 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2821 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2881 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2941 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3001 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     3061 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3121 ccgctacagg gcgcgtgggg atacccccta gagccccagc tggttctttc cgcctcagaa
     3181 gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttccccttg ctgtcctgcc
     3241 ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat
     3301 tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg
     3361 ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag cgggtttaaa
     3421 cgggccctct agactcgagt tatcacttgt catcgtcatc cttgtagtcg atgtcatgat
     3481 ctttataatc accgtcatgg tctttgtaat caaccacttt gtacaagaaa gctgggtcgc
     3541 cgttgaacat tattgaagcg gggtcttggt ttgccgtctg ggccatatgc cggaggatat
     3601 ctttttttgt tataatgcca aggaggcgcc cattgtgagt tacaaggcac tgcctcagtc
     3661 ccagctttcg gaaaatatcc accacgatct ccattggggt gtggtctgtc actgtaaaag
     3721 ggctcatgtc aagaatgctt cgaagcttca atggccgagg actttctgct ggaagagatg
     3781 gggtgtgctg tgcaaaacac acccgagaac tgccaacgat accttctcgt tttttcctgg
     3841 cactttctat tgcaattgtc aggtctcttc tgagggcaaa tcccactaat ctctgagatt
     3901 cttttgacat tatgacagga aatccattgt agctggtttc attaatcatg ttttctatat
     3961 catccactgt catattgtcc tgtgtcagga cagctaaggg aggatcattc cttcgaggtc
     4021 tcataacgtc agcagccagg gtggtatgag tgaattcttc ttttgcatcc aagaaggggt
     4081 atccatttaa tcggatgtgt gcttcataaa tgccttccct gccaaaggca tctccaaccc
     4141 atttactggt catgactgca gccataaggg gaacaatata ttccaagcct ccagtaagct
     4201 caaaaacaat aaccaccagg gagacagtca ttcttgtcac accacctaag catgcagcag
     4261 caccaaccat ggcataaagg ccaggtgcaa tgcaatcagc cccgacctca caccactcct
     4321 taaagataaa ccagtcgtgg tgacagtagg caagctgctc caccgcaatc cccacaatcc
     4381 ttcctgcgat cgctccaatg gccatgctgg ggatgaacaa gcctgatgga accttgatgc
     4441 caaaagtgaa tactgtcatt atgattttaa atatgagtgc caggcataac tgccatatag
     4501 ctgaatatac tccaatgcct gctggacgat caggaatgtc atcgacaatt ttactggcat
     4561 tcatgtcatt tctgtagtca caaagagaag aggattccag gggaccacag tctgtaaaaa
     4621 gctctttgat cagttcactg gtgtttagcc tagtgtatgg attagggaag gctatcacag
     4681 cagtaatggc tgcaacaata atgacttcca gaacgggata ctttccaaat ttcgtggact
     4741 tgcgtcgacg acaccaggca atatttgccc taatgaaaaa ggctccccaa agccctccaa
     4801 atacccctag aagaataaaa ggaaacagtt caaaaaggta ccatggtgta tgatactcca
     4861 cataaaaaag gaccagacgg ctgttaccaa atggattgat ggacctcaaa acaaatgcag
     4921 ccactaaagc agcaaaaaat gatctccata aagttttgag aggaaaataa tagctaacct
     4981 cttccaggct aaaaagaact cctccaattg gtgcaccaaa agctacagaa acccctgcag
     5041 ctgaggcagc tgatagcacc tccctttttt tagcttcgtt tgtgctatac tttggaaaga
     5101 ggtaggaaaa gatatttccg cagcaacagg caacatgtac caggggacct tcttttccta
     5161 aactcaaacc tgatgccaca gccaggacta atgtgatggt tttaatcatt aaagtccatt
     5221 ttcccaagta acctctgatg atgaatccac ttaaaatagt tttaatctct ggaattccag
     5281 agccacaggc atatggagca aataccttta ccagggaaac tgcaagaaag gcaaaactca
     5341 aggcccagaa gatgtacatt atgtagttcg tgatataaga accaggaccc tctgcttgac
     5401 ctatgattaa ttctgcccat gttttccact gtggacattt atccctctct tcaaatgttg
     5461 tttcattaga tccccaacag cactgttcgt ggttgtacca caacgcacta aggcaaatgc
     5521 cctcctttag gtcagtcatc caatcggcag caatgtctat taatccggcc agtgcccctg
     5581 atgccaatcc tgttagtgtt actactagcc atcctgacca cgcatcatac aaactttttg
     5641 tcatttccca tgctgattct ttctttttgc tgttgatccg tctatgcctt tctctgtctt
     5701 tacatttttc tcgcacccaa tcaatagtat ggaaatcatc atatgtacca acacctggaa
     5761 ttggttcatc caaaagatcc agtaaatgtg tagaactgtt aatgctgcct ccatttgtca
     5821 ttgtataatg agttccaact gcagtgtcac catctaataa attgtcatct tcagatgttt
     5881 gaaagtccat aataacacct gctccatcta aaagttcctc atcactactt gcacttgtta
     5941 tactgttgta gctgtttcta tagtagcctc tatggaacag ctgctcagac tccatggtgc
     6001 cagctttttt gtacaaactt gttgatatct gcagaattcc accacactgg actagtggat
     6061 ccgagctcgg taccaagctt aagtttaaac gctagccagc ttgggtctcc ctatagtgag
     6121 tcgtattaat ttcgataagc cagtaagcag tgggttctct agttagccag agagctctgc
     6181 ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt
     6241 tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa
     6301 tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact
     6361 gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag
     6421 gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga
     6481 cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt
     6541 accgtaaata ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa
     6601 catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat
     6661 ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa
     6721 ttgattacta ttaataacta gtcaataatc aatgtcaacg cgtatatctg gcccgtacat
     6781 cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt catgcaattg tcggtcaagc
     6841 cttgccttgt tgtagcttaa attttgctcg cgcactactc agcgacctcc aacacacaag
     6901 cagggagcag atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     6961 ataggggatc gggagatctc ccgatccgtc gacgtcaggt ggcacttttc ggggaaatgt
     7021 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     7081 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     7141 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     7201 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     7261 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     7321 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     7381 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     7441 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     7501 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     7561 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     7621 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     7681 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     7741 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     7801 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     7861 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     7921 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     7981 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     8041 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     8101 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
