LOCUS       sequence-348257-        7086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Nhx1-HaloTagx2  in yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7086)
  AUTHORS   Casler JC, Johnson N, Krahn AH, Pantazopoulou A, Day KJ, Glick BS
  TITLE     Clathrin adaptors mediate two sequential pathways of intra-Golgi 
            recycling.
  JOURNAL   J Cell Biol. 2022 Jan 3;221(1). pii: 212747. doi: 
            10.1083/jcb.202103199. Epub 2021 Nov 5.
  PUBMED    34739034
REFERENCE   2  (bases 1 to 7086)
  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 
            Biol."; date: "2022-01-3"; volume: "221(1). pii: 212747. doi"; 
            pages: " 10.1083/jcb.202103199. Epub 2021 Nov 5"
COMMENT     Sequence Label: YIplac211-NHX1-HaloTagx2
FEATURES             Location/Qualifiers
     source          1..7086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1037..1927
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1955..2845
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     primer_bind     complement(3584..3601)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3584..3600)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3593..3615)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3809..3828)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3928..3950
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4068..4871)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4872..5087)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5094..5112)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5180..5284
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5285..5353,5354..6145)
                     /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(5503..5522)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6316..6904
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6805..6824
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7058..7075
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
      241 atgcgcagct atttctatat gtgttgctcg ttggtgtgct gtgttcccat tgtcgcaatt
      301 tgttaactgg atatatagag taaagacaat cagatctatg agcggcataa ccggagaaaa
      361 tatttctgtt cccgatgaaa taccctacaa ttaccaaatg atgacatttt gggcaggttt
      421 acgtggtgct gttggtgtcg ccttggcgtt gggaattcaa ggtgagtata agttcacttt
      481 attggcaacg gtccttgttg ttgttgtttt aacagttatc atttttgggg gcactactgc
      541 aggaatgtta gaagttttaa atattaagac tggttgcata agtgaagaag atacatctga
      601 tgacgagttt gatatagagg ctccaagggc gataaattta ttgaacggta gttctattca
      661 gacagatttg ggcccatatt ctgacaacaa ttctccagat atttcaattg accaattcgc
      721 ggtcagcagt aacaagaatc tccccaataa catatccaca actggtggta atacttttgg
      781 aggccttaat gaaactgaga atactagccc taacccggca aggtcttcta tggataagcg
      841 taatttgaga gataaactgg gaacaatctt taattccgac tcacaatggt ttcaaaattt
      901 tgatgaacag gtattgaagc cagtattctt ggacaacgtt tctccatcct tacaagattc
      961 ggctacgcaa tcacctgcag atttctcttc ccaaaaccac acggatccgg ctagcggagg
     1021 aagttcgggg agttcgatgg cagaaatcgg tactggcttt ccattcgacc cccattatgt
     1081 ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt
     1141 gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt
     1201 tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc
     1261 agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct
     1321 gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg
     1381 ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat
     1441 cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct tccgcaccac
     1501 cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta cgctgccgat
     1561 gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa
     1621 tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc
     1681 agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc
     1741 gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct
     1801 ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccgggtctga atctgctgca
     1861 agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcga cgctcgagat
     1921 ttccggctct agcggaggaa gttcggggag ttcgatggca gaaatcggta ctggctttcc
     1981 attcgacccc cattatgtgg aagtcctggg cgagcgcatg cactacgtcg atgttggtcc
     2041 gcgcgatggc acccctgtgc tgttcctgca cggtaacccg acctcctcct acgtgtggcg
     2101 caacatcatc ccgcatgttg caccgaccca tcgctgcatt gctccagacc tgatcggtat
     2161 gggcaaatcc gacaaaccag acctgggtta tttcttcgac gaccacgtcc gcttcatgga
     2221 tgccttcatc gaagccctgg gtctggaaga ggtcgtcctg gtcattcacg actggggctc
     2281 cgctctgggt ttccactggg ccaagcgcaa tccagagcgc gtcaaaggta ttgcatttat
     2341 ggagttcatc cgccctatcc cgacctggga cgaatggcca gaatttgccc gcgagacctt
     2401 ccaggccttc cgcaccaccg acgtcggccg caagctgatc atcgatcaga acgtttttat
     2461 cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact gaagtcgaga tggaccatta
     2521 ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg tggcgcttcc caaacgagct
     2581 gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc gaagaataca tggactggct
     2641 gcaccagtcc cctgtcccga agctgctgtt ctggggcacc ccaggcgttc tgatcccacc
     2701 ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc aaggctgtgg acatcggccc
     2761 gggtctgaat ctgctgcaag aagacaaccc ggacctgatc ggcagcgaga tcgcgcgctg
     2821 gctgtcgacg ctcgagattt ccggctctag ataagcggcc gcactttaaa gtgtatggtt
     2881 tccttgtttc taatataaat atattaatta agtctttata cattctctct tactcaacgc
     2941 cgaaggaata cccttctaca ctttattcat tttaaatcat gtactgttat atacacgcta
     3001 gtaggattga tcttatttaa tcatatttcg cacgttaccc gactgctttc agttttgccg
     3061 ctttgtgata atgaggaatt tcagctagct tttaatgagc cgatactaaa tcaccatgat
     3121 tttgggagag gaaaagaaga aaaggtaaaa caacgaatat ttttcaaata ccttagttac
     3181 tgaatatcca taactactcg tataattggt gagaaaatag tgcatattta gtttactttt
     3241 tgcctttgat tgaaaatata tattcatggt gctttttact cggtgtgaaa aggcaagaaa
     3301 ggagaaactc gccgctggtt ataagccctt agttgattat ttgattgatt gtgatactcc
     3361 cacattttta gaaagaattg aggcaatcca agagtgggac cgatccagag atgatcttta
     3421 tgtttggatt cccattctag acagaatgga cgggctgttg ttaaaagtgg cagagaaata
     3481 taaatacaag caagacccca agaaggagtg tgaagtcaaa ttggtggaaa tggaagcgca
     3541 tgatgttgat tattgtttga aaatgggtac cgagctcgaa ttcactggcc gtcgttttac
     3601 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     3661 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     3721 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3781 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3841 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3901 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3961 catcaccgaa acgcgcgaga cgaaagggcg ggtaataact gatataatta aattgaagct
     4021 ctaatttgtg agtttagtat acatgcattt acttataata cagtttttta gttttgctgg
     4081 ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc ctctacctta
     4141 gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga tcctgtagag
     4201 accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc atctaaaccc
     4261 acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat gtctctttga
     4321 gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt acccttagta
     4381 tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa aaggcctcta
     4441 ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc
     4501 acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc cgcagagtac
     4561 tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag taaaaaattg
     4621 tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa atcagtcaag
     4681 atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac taactccagt
     4741 aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg
     4801 atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc cttatatgta
     4861 gctttcgaca tgatttatct tcgtttcctg catgtttttg ttctgtgcag ttgggttaag
     4921 aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac caatctaagt
     4981 ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa
     5041 ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaaaag ctggcctcgt
     5101 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     5161 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5221 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5281 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5341 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5401 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5461 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5521 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5581 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5641 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5701 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5761 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5821 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5881 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5941 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6001 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6061 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6121 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6181 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6241 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6301 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6361 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6421 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6481 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6541 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6601 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6661 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6721 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6781 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6841 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6901 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6961 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     7021 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     7081 ggaaga
//
