LOCUS       sequence-348208-        5484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Ent5-HaloTag in yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5484)
  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 5484)
  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-ENT5-HaloTag
FEATURES             Location/Qualifiers
     source          1..5484
                     /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             complement(286..303)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             567..1457
                     /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(1982..1999)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1982..1998)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1991..2013)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2207..2226)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2326..2348
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2466..3269)
                     /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(3270..3485)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3492..3510)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3578..3682
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3683..3751,3752..4543)
                     /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(3901..3920)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4714..5302
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5203..5222
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5456..5473
                     /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 atgctggtaa taatgacaac aaaaacaata atatcaacag caatagtgat gatgatgatg
      301 atgagtttgg cgacttccaa agcgaaacga gtcccgacac aacagcacct aaaacttcca
      361 atagtaaaat agatgatttg cttgactggg atggcccaaa gtcagacaca gataccaccg
      421 ctgctgcgca aacatcattg ccctttgcgg agaaaaaaca gcaaaaagcc cgtcctcagg
      481 ctacaaagga caagtcgaag ggaaatgatg cattttccga cttattctct tactccaagt
      541 cgttggtcgg cagcagtggt agtagcatgg cagaaatcgg tacaggtttc ccttttgacc
      601 cacactatgt cgaagtatta ggtgaaagaa tgcactatgt tgatgtcggt cctagagatg
      661 gtactccagt tttgttttta catggtaacc caacatcttc atacgtttgg agaaacatca
      721 tcccacatgt tgcaccaact catagatgta ttgctccaga tttgattggt atgggtaaat
      781 ctgataagcc agatttgggt tatttctttg atgatcatgt tagattcatg gatgctttta
      841 ttgaagcatt gggtttggaa gaagttgttt tggttattca tgattggggt tcagcattag
      901 gttttcattg ggctaagaga aacccagaaa gagttaaggg tatcgctttt atggaattca
      961 ttagaccaat tccaacttgg gatgaatggc cagaattcgc aagagaaaca ttccaagctt
     1021 ttagaactac agatgttggt agaaagttga tcatcgatca aaacgttttt attgaaggta
     1081 ctttgccaat gggtgttgtt agaccattaa cagaagttga aatggatcat tacagagaac
     1141 catttttaaa cccagttgat agagaaccat tgtggagatt tccaaatgaa ttaccaattg
     1201 ctggtgaacc agcaaacatc gttgctttgg ttgaagaata catggattgg ttacatcaat
     1261 ctccagttcc aaagttgtta ttttggggta caccaggtgt tttgattcca ccagcagaag
     1321 ctgcaagatt ggctaagtca ttgccaaact gtaaggctgt tgatattggt ccaggtttaa
     1381 atttgttaca agaagataat ccagacttga taggtagtga aatagccaga tggttgtcca
     1441 cattagaaat ctcaggttaa tcaccaaata ccccttcatc tcccaataca ttcaatacag
     1501 tcaacaaatt ttcattctca gatatatgta tccatatata cactaataat atcttactaa
     1561 aaacttgtct atttccttct tcaaatgtca gttttctttt tattgaatat caaaaaaata
     1621 ttatatagat acatttgtat atgtcattat ccatgcagaa gaaaatactt aaatacttaa
     1681 gtacacaagt atttattgat ttgctatttc ggtcttggca agtcccttgg ctacgtcatc
     1741 tacactcgcg tcatcctctt ccatatactc gtcgcctgtg cccaccacgc cttgttcaaa
     1801 catttgctta ccggttagct tcccatgatg agccttcatc cttctcacct ggtctctttc
     1861 atccagtttc agttcttggc gaaatttaga cctccattcc aagtacgatt ctcttgtgac
     1921 tttagtacca tggaacttgg cctgttcctt cttttcacgt tctggtaccg agctcgaatt
     1981 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     2041 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     2101 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc
     2161 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     2221 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     2281 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     2341 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcggg taataactga
     2401 tataattaaa ttgaagctct aatttgtgag tttagtatac atgcatttac ttataataca
     2461 gttttttagt tttgctggcc gcatcttctc aaatatgctt cccagcctgc ttttctgtaa
     2521 cgttcaccct ctaccttagc atcccttccc tttgcaaata gtcctcttcc aacaataata
     2581 atgtcagatc ctgtagagac cacatcatcc acggttctat actgttgacc caatgcgtct
     2641 cccttgtcat ctaaacccac accgggtgtc ataatcaacc aatcgtaacc ttcatctctt
     2701 ccacccatgt ctctttgagc aataaagccg ataacaaaat ctttgtcgct cttcgcaatg
     2761 tcaacagtac ccttagtata ttctccagta gatagggagc ccttgcatga caattctgct
     2821 aacatcaaaa ggcctctagg ttcctttgtt acttcttctg ccgcctgctt caaaccgcta
     2881 acaatacctg ggcccaccac accgtgtgca ttcgtaatgt ctgcccattc tgctattctg
     2941 tatacacccg cagagtactg caatttgact gtattaccaa tgtcagcaaa ttttctgtct
     3001 tcgaagagta aaaaattgta cttggcggat aatgccttta gcggcttaac tgtgccctcc
     3061 atggaaaaat cagtcaagat atccacatgt gtttttagta aacaaatttt gggacctaat
     3121 gcttcaacta actccagtaa ttccttggtg gtacgaacat ccaatgaagc acacaagttt
     3181 gtttgctttt cgtgcatgat attaaatagc ttggcagcaa caggactagg atgagtagca
     3241 gcacgttcct tatatgtagc tttcgacatg atttatcttc gtttcctgca tgtttttgtt
     3301 ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt cttcaacact acatatgcgt
     3361 atatatacca atctaagtct gtgctccttc cttcgttctt ccttctgttc ggagattacc
     3421 gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag aataaaaaaa aaatgatgaa
     3481 ttgaaaagct ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     3541 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3601 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3661 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3721 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3781 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3841 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3901 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3961 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4021 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4081 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4141 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4201 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     4261 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     4321 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4381 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4441 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4501 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4561 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4621 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4681 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4741 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4801 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4861 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4921 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4981 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5041 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5101 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5161 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5221 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5281 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5341 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5401 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     5461 gtcagtgagc gaggaagcgg aaga
//
