LOCUS       sequence-222004-        5878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Sec31-yoHalo at the endogenous locus via pop-in pop-out 
            mutagenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5878)
  AUTHORS   Casler JC, Papanikou E, Barrero JJ, Glick BS
  TITLE     Maturation-driven transport and AP-1-dependent recycling of a 
            secretory cargo in the Golgi.
  JOURNAL   J Cell Biol. 2019 May 6;218(5):1582-1601. doi: 
            10.1083/jcb.201807195. Epub 2019 Mar 11.
  PUBMED    30858194
REFERENCE   2  (bases 1 to 5878)
  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: 
            "10.1083/jcb.201807195"; journalName: "J Cell Biol"; date: 
            "2019-05-6- 6"; volume: "218"; issue: "5"; pages: "1582-1601"
COMMENT     Sequence Label: YIplac211-Sec31-yoHalo
FEATURES             Location/Qualifiers
     source          1..5878
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    77..98
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(113..118,119..136,137..143)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    151..167
                     /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     156..178
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     175..191
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     175..191
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             847..1737
                     /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(2327..2344)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2327..2343)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2336..2358)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2552..2571)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2671..2693
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2731..2749)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2910..3125
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3126..3926
                     /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
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQLN"
     CDS             join(4048..4116,4117..4908)
                     /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(4266..4285)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5079..5667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5568..5587
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5821..5838
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
       61 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
      121 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
      181 acagctatga ccatgattac gccaagctcc tctaatccat atgctccaac tgcaaccact
      241 caacccaacg gttcctccta tcctccaacc ggtccgtata ctaataacca taccatgacc
      301 tctcctcctc ccgtttttaa caaacctccc actggccccc ctccgattag catgaagaag
      361 agaagcaaca agttagctag tatagaacaa aacccatctc aaggtgctac ttatcctcca
      421 accctttcca gctcggcctc tccattgcag ccttctcaac cgccaacttt ggcttctcag
      481 gttaatacct ccgctgagaa tgtcagtcat gaaattccag ctgatcaaca acccattgtc
      541 gacttcttga aagaagaact ggctcgcgta acaccattga ccccaaagga gtactccaaa
      601 caattaaagg attgtgataa acgattaaag attcttttct accatttgga aaagcaggat
      661 ttattaaccc aaccaacaat cgattgttta catgacctcg tcgcattaat gaaggaaaag
      721 aaatacaaag aagctatggt catccatgct aatatcgcta caaaccatgc tcaagagggt
      781 ggtaactggc tgacaggagt gaagaggttg attggcatag ctgaagcgac tttgaattac
      841 gatcccatgg cagaaatcgg tacaggtttc ccttttgacc cacactatgt cgaagtatta
      901 ggtgaaagaa tgcactatgt tgatgtcggt cctagagatg gtactccagt tttgttttta
      961 catggtaacc caacatcttc atacgtttgg agaaacatca tcccacatgt tgcaccaact
     1021 catagatgta ttgctccaga tttgattggt atgggtaaat ctgataagcc agatttgggt
     1081 tatttctttg atgatcatgt tagattcatg gatgctttta ttgaagcatt gggtttggaa
     1141 gaagttgttt tggttattca tgattggggt tcagcattag gttttcattg ggctaagaga
     1201 aacccagaaa gagttaaggg tatcgctttt atggaattca ttagaccaat tccaacttgg
     1261 gatgaatggc cagaattcgc aagagaaaca ttccaagctt ttagaactac agatgttggt
     1321 agaaagttga tcatcgatca aaacgttttt attgaaggta ctttgccaat gggtgttgtt
     1381 agaccattaa cagaagttga aatggatcat tacagagaac catttttaaa cccagttgat
     1441 agagaaccat tgtggagatt tccaaatgaa ttaccaattg ctggtgaacc agcaaacatc
     1501 gttgctttgg ttgaagaata catggattgg ttacatcaat ctccagttcc aaagttgtta
     1561 ttttggggta caccaggtgt tttgattcca ccagcagaag ctgcaagatt ggctaagtca
     1621 ttgccaaact gtaaggctgt tgatattggt ccaggtttaa atttgttaca agaagataat
     1681 ccagacttga taggtagtga aatagccaga tggttgtcca cattagaaat ctcaggttaa
     1741 agcggccgta agtctcaaaa ctttcagtac aaaaagtggc gtattggcct tgtttttttc
     1801 ttattatgtc ttatagtatt atatttatat atttatgtac ttatcctttt atgtttaaac
     1861 ctaatcaaga tcacaattag aactagctta acattacttc tcaaaaccaa ccaatacctc
     1921 ttatatcttt tttacagcct ttcccttatg tttttaccat gtacacctcg agcggcaaat
     1981 ctccggggaa attttaaact tgccgcatcg cacattctaa agaacaaaag gaggaggaac
     2041 tgtcaattat tctataatct ggggaaattc agtcatactg agaaaaacta gacaatagtc
     2101 ctatcctcgg caaattgcat tgatagtact tacacatcga gagatcatag gacagataag
     2161 aagcatttta ttgaagaaaa acagagagag tcactataag ttgacctcac tatagaaatt
     2221 aaaacagaaa agttttactg aggacctttt taaacagaag gatatgcctt tgttggcata
     2281 gaaagaagaa tttataaatg gatccccggg taccgagctc gaattcactg gccgtcgttt
     2341 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2401 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2461 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg
     2521 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     2581 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     2641 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     2701 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     2761 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     2821 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2881 aaccctgata aatgcttcaa taatagcttt tcaattcatc attttttttt tattcttttt
     2941 tttgatttcg gtttccttga aatttttttg attcggtaat ctccgaacag aaggaagaac
     3001 gaaggaagga gcacagactt agattggtat atatacgcat atgtagtgtt gaagaaacat
     3061 gaaattgccc agtattctta acccaactgc acagaacaaa aacctgcagg aaacgaagat
     3121 aaatcatgtc gaaagctaca tataaggaac gtgctgctac tcatcctagt cctgttgctg
     3181 ccaagctatt taatatcatg cacgaaaagc aaacaaactt gtgtgcttca ttggatgttc
     3241 gtaccaccaa ggaattactg gagttagttg aagcattagg tcccaaaatt tgtttactaa
     3301 aaacacatgt ggatatcttg actgattttt ccatggaggg cacagttaag ccgctaaagg
     3361 cattatccgc caagtacaat tttttactct tcgaagacag aaaatttgct gacattggta
     3421 atacagtcaa attgcagtac tctgcgggtg tatacagaat agcagaatgg gcagacatta
     3481 cgaatgcaca cggtgtggtg ggcccaggta ttgttagcgg tttgaagcag gcggcagaag
     3541 aagtaacaaa ggaacctaga ggccttttga tgttagcaga attgtcatgc aagggctccc
     3601 tatctactgg agaatatact aagggtactg ttgacattgc gaagagcgac aaagattttg
     3661 ttatcggctt tattgctcaa agagacatgg gtggaagaga tgaaggttac gattggttga
     3721 ttatgacacc cggtgtgggt ttagatgaca agggagacgc attgggtcaa cagtatagaa
     3781 ccgtggatga tgtggtctct acaggatctg acattattat tgttggaaga ggactatttg
     3841 caaagggaag ggatgctaag gtagagggtg aacgttacag aaaagcaggc tgggaagcat
     3901 atttgagaag atgcggccag ctaaactcac aaattagagc ttcaatttaa ttatatcagt
     3961 tattacccgg gaatctcggt cgtaatgatt tttataatga cgaaaaaaaa aaaattggaa
     4021 agaaaaagct attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4081 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4141 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4201 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4261 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4321 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4381 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4441 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4501 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4561 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     4621 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4681 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4741 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4801 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4861 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4921 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4981 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5041 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5101 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5161 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5221 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5281 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5341 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5401 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5461 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5521 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5581 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5641 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5701 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5761 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5821 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccg
//
