LOCUS       sequence-202561-        6118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for pop-in/pop-out replacement of Tlg1 with yoHaloTag-Tlg1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6118)
  AUTHORS   Day KJ, Casler JC, Glick BS
  TITLE     Budding Yeast Has a Minimal Endomembrane System.
  JOURNAL   Dev Cell. 2018 Jan 8;44(1):56-72.e4. doi: 
            10.1016/j.devcel.2017.12.014. Epub 2018 Jan 8.
  PUBMED    29316441
REFERENCE   2  (bases 1 to 6118)
  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: 
            "10.1016/j.devcel.2017.12.014"; journalName: "Dev Cell"; date: 
            "2018-01-8- 8"; volume: "44"; issue: "1"; pages: "56-72.e4"
COMMENT     Sequence Label: YIplac211-yoHaloTag-TLG1
FEATURES             Location/Qualifiers
     source          1..6118
                     /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             670..1560
                     /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(2616..2633)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2616..2632)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2625..2647)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2841..2860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2960..2982
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3100..3903)
                     /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(3904..4119)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4126..4144)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4212..4316
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4317..4385,4386..5177)
                     /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(4535..4554)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5348..5936
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5837..5856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6090..6107
                     /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 atgcttcgcg ttgaatttgc tccattggaa tatcagcatt acttgaagta tcctctacca
      301 taggaaccgt aacttcatta tgttggggac tattctcact ttcattcatt gtataggttt
      361 ccctaattct cttaagagaa cctgcttatg aaggcttttt tttgtttgga tcaccatctg
      421 tttttctttt tttatcgagt ttttaattat ccacgagata ttcgaaccta cgacataaag
      481 tagtaaatat cagaagatag gacaattcga agcacacaac acagaacata tactaaaatt
      541 atatttgctg tctttctgac aaggtttgtg aacattttaa tttctacgtt ttccctgtaa
      601 ctgaagtaaa cgtgaaagac gtttaactgt aaaattgtat tgctaagatc ctttctttaa
      661 caaatgaaca tggctgaaat cggtacaggt tttccttttg acccacacta tgtcgaagta
      721 ttaggtgaaa gaatgcacta tgttgatgtc ggtcctagag atggtactcc agttttgttt
      781 ttacatggta acccaacatc ttcatacgtt tggagaaaca tcatcccaca tgttgcacca
      841 actcatagat gtattgctcc agatttgatt ggtatgggta aatctgataa gccagatttg
      901 ggttatttct ttgatgatca tgttagattc atggatgctt ttattgaagc attgggtttg
      961 gaagaagttg ttttggttat tcatgattgg ggttcagcat taggttttca ttgggctaag
     1021 agaaacccag aaagagttaa gggtatcgct tttatggaat tcattagacc aattccaact
     1081 tgggatgaat ggccagaatt cgcaagagaa acattccaag cttttagaac tacagatgtt
     1141 ggtagaaagt tgatcatcga tcaaaacgtt tttattgaag gtactttgcc aatgggtgtt
     1201 gttagaccat taacagaagt tgaaatggat cattacagag aaccattttt aaacccagtt
     1261 gatagagaac cattgtggag atttccaaat gaattaccaa ttgctggtga accagcaaac
     1321 atcgttgctt tggttgaaga atacatggat tggttacatc aatctccagt tccaaagttg
     1381 ttattttggg gtacaccagg tgttttgatt ccaccagcag aagctgcaag attggctaag
     1441 tcattgccaa actgtaaggc tgttgatatt ggtccaggtt taaatttgtt acaagaagat
     1501 aatccagact taataggttc cgaaatcgcc agatggttat caactttaga aatcagtggt
     1561 aacagtgaag atccgtttca acaagttgtt aaggacacca aggagcaatt gaaccgcata
     1621 aacaattaca taactcgtca caatactgct ggtgatgacg atcaagagga ggaaatacaa
     1681 gatattttaa aggatgttga ggaaacaata gttgatttgg acagaagcat aatcgtaatg
     1741 aaaagggatg aaaacgaaga cgtgagtggt agggaagcac aagttaaaaa tataaaacag
     1801 caacttgatg ctctgaagtt gcgttttgat cgaagaatac aggaatctac tcaaacaact
     1861 attcctctag aagagacggt ggaaaattca acacttaaca ccagcatggc tgagaacaat
     1921 gatggtggta tgtccaatcc gtttcaggaa caaatgttaa gagaacaaga tgttcattta
     1981 gatggtattc acaagacaat gcaaaatttg catattcaag ctcaaacaat gggggatgaa
     2041 ttagagaacc agggacaatt gttggataat atggacgagg gtatggacgg tgttgtaaat
     2101 aagctggcta gaggtcgtag gcaattggaa tgggtctacg aaaaaaataa agaaaaatac
     2161 gacgattgtt gtataggact tcttattgtc gtcttgatag ttttattagt tttggcattc
     2221 attgcttgaa aaatacaatg cactaatgcc gaagagcttc aatcgaagat atactgggca
     2281 tatataaaat acatgcattc tgaatacata agtgatacgg atattaatgc tacaagggtt
     2341 tggatatgat ttatatgttg gtttttctgg atcgtcaaca actcgtgcat atttttttgt
     2401 ttcgaagtag aggaaccaca aatattatta aagcgtgtta tttttttttt cttactcttc
     2461 tgtaaacttc taaatgctgc tgaggcaccc ctactagcaa tttccctatc aaagtttgtt
     2521 tggctgttat taagggtatg taagacctcg tatctgctgc tactgatcac catgttagaa
     2581 ggagcagaat acttttctct cgtagttggg aattcactgg ccgtcgtttt acaacgtcgt
     2641 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2701 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2761 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2821 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     2881 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2941 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3001 aaacgcgcga gacgaaaggg cgggtaataa ctgatataat taaattgaag ctctaatttg
     3061 tgagtttagt atacatgcat ttacttataa tacagttttt tagttttgct ggccgcatct
     3121 tctcaaatat gcttcccagc ctgcttttct gtaacgttca ccctctacct tagcatccct
     3181 tccctttgca aatagtcctc ttccaacaat aataatgtca gatcctgtag agaccacatc
     3241 atccacggtt ctatactgtt gacccaatgc gtctcccttg tcatctaaac ccacaccggg
     3301 tgtcataatc aaccaatcgt aaccttcatc tcttccaccc atgtctcttt gagcaataaa
     3361 gccgataaca aaatctttgt cgctcttcgc aatgtcaaca gtacccttag tatattctcc
     3421 agtagatagg gagcccttgc atgacaattc tgctaacatc aaaaggcctc taggttcctt
     3481 tgttacttct tctgccgcct gcttcaaacc gctaacaata cctgggccca ccacaccgtg
     3541 tgcattcgta atgtctgccc attctgctat tctgtataca cccgcagagt actgcaattt
     3601 gactgtatta ccaatgtcag caaattttct gtcttcgaag agtaaaaaat tgtacttggc
     3661 ggataatgcc tttagcggct taactgtgcc ctccatcgaa aaatcagtca agatatccac
     3721 atgtgttttt agtaaacaaa ttttgggacc taatgcttca actaactcca gtaattcctt
     3781 ggtggtacga acatccaatg aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa
     3841 tagcttggca gcaacaggac taggatgagt agcagcacgt tccttatatg tagctttcga
     3901 catgatttat cttcgtttcc tgcatgtttt tgttctgtgc agttgggtta agaatactgg
     3961 gcaatttcat gtttcttcaa cactacatat gcgtatatat accaatctaa gtctgtgctc
     4021 cttccttcgt tcttccttct gttcggagat taccgaatca aaaaaatttc aaggaaaccg
     4081 aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaaa agctggcctc gtgatacgcc
     4141 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     4201 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4261 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4321 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4381 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     4441 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4501 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4561 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     4621 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4681 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     4741 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4801 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4861 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4921 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4981 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5041 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5101 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5161 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5221 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5281 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5341 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5401 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5461 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5521 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5581 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5641 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5701 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5761 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5821 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5881 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5941 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6001 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6061 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaaga
//
