LOCUS       sequence-217495-        5918 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 5918)
  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 5918)
  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..5918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    75..96
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(111..116,117..134,135..141)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    149..165
                     /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     154..176
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     173..189
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     173..189
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             845..1735
                     /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(2325..2342)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2325..2341)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2334..2356)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2550..2569)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2669..2691
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2729..2747)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2908..3124
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3125..3928
                     /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
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLATGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     CDS             join(4086..4154,4155..4946)
                     /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(4304..4323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5117..5705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5606..5625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5859..5876
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
       61 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
      121 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      181 agctatgacc atgattacgc caagctcctc taatccatat gctccaactg caaccactca
      241 acccaacggt tcctcctatc ctccaaccgg tccgtatact aataaccata ccatgacctc
      301 tcctcctccc gtttttaaca aacctcccac tggcccccct ccgattagca tgaagaagag
      361 aagcaacaag ttagctagta tagaacaaaa cccatctcaa ggtgctactt atcctccaac
      421 cctttccagc tcggcctctc cattgcagcc ttctcaaccg ccaactttgg cttctcaggt
      481 taatacctcc gctgagaatg tcagtcatga aattccagct gatcaacaac ccattgtcga
      541 cttcttgaaa gaagaactgg ctcgcgtaac accattgacc ccaaaggagt actccaaaca
      601 attaaaggat tgtgataaac gattaaagat tcttttctac catttggaaa agcaggattt
      661 attaacccaa ccaacaatcg attgtttaca tgacctcgtc gcattaatga aggaaaagaa
      721 atacaaagaa gctatggtca tccatgctaa tatcgctaca aaccatgctc aagagggtgg
      781 taactggctg acaggagtga agaggttgat tggcatagct gaagcgactt tgaattacga
      841 tcccatggca gaaatcggta caggtttccc ttttgaccca cactatgtcg aagtattagg
      901 tgaaagaatg cactatgttg atgtcggtcc tagagatggt actccagttt tgtttttaca
      961 tggtaaccca acatcttcat acgtttggag aaacatcatc ccacatgttg caccaactca
     1021 tagatgtatt gctccagatt tgattggtat gggtaaatct gataagccag atttgggtta
     1081 tttctttgat gatcatgtta gattcatgga tgcttttatt gaagcattgg gtttggaaga
     1141 agttgttttg gttattcatg attggggttc agcattaggt tttcattggg ctaagagaaa
     1201 cccagaaaga gttaagggta tcgcttttat ggaattcatt agaccaattc caacttggga
     1261 tgaatggcca gaattcgcaa gagaaacatt ccaagctttt agaactacag atgttggtag
     1321 aaagttgatc atcgatcaaa acgtttttat tgaaggtact ttgccaatgg gtgttgttag
     1381 accattaaca gaagttgaaa tggatcatta cagagaacca tttttaaacc cagttgatag
     1441 agaaccattg tggagatttc caaatgaatt accaattgct ggtgaaccag caaacatcgt
     1501 tgctttggtt gaagaataca tggattggtt acatcaatct ccagttccaa agttgttatt
     1561 ttggggtaca ccaggtgttt tgattccacc agcagaagct gcaagattgg ctaagtcatt
     1621 gccaaactgt aaggctgttg atattggtcc aggtttaaat ttgttacaag aagataatcc
     1681 agacttgata ggtagtgaaa tagccagatg gttgtccaca ttagaaatct caggttaaag
     1741 cggccgtaag tctcaaaact ttcagtacaa aaagtggcgt attggccttg tttttttctt
     1801 attatgtctt atagtattat atttatatat ttatgtactt atccttttat gtttaaacct
     1861 aatcaagatc acaattagaa ctagcttaac attacttctc aaaaccaacc aatacctctt
     1921 atatcttttt tacagccttt cccttatgtt tttaccatgt acacctcgag cggcaaatct
     1981 ccggggaaat tttaaacttg ccgcatcgca cattctaaag aacaaaagga ggaggaactg
     2041 tcaattattc tataatctgg ggaaattcag tcatactgag aaaaactaga caatagtcct
     2101 atcctcggca aattgcattg atagtactta cacatcgaga gatcatagga cagataagaa
     2161 gcattttatt gaagaaaaac agagagagtc actataagtt gacctcacta tagaaattaa
     2221 aacagaaaag ttttactgag gaccttttta aacagaagga tatgcctttg ttggcataga
     2281 aagaagaatt tataaatgga tccccgggta ccgagctcga attcactggc cgtcgtttta
     2341 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     2401 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2461 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2521 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2581 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     2641 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     2701 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     2761 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2821 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2881 ccctgataaa tgcttcaata atagcttttc aattcatctt tttttttttt gttctttttt
     2941 ttgattccgg tttctttgaa atttttttga ttcggtaatc tccgagcaga aggaagaacg
     3001 aaggaaggag cacagactta gattggtata tatacgcata tgtggtgttg aagaaacatg
     3061 aaattgccca gtattcttaa cccaactgca cagaacaaaa acctgcagga aacgaagata
     3121 aatcatgtcg aaagctacat ataaggaacg tgctgctact catcctagtc ctgttgctgc
     3181 caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat tggatgttcg
     3241 taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt gtttactaaa
     3301 aacacatgtg gatatcttga ctgatttttc catggagggc acagttaagc cgctaaaggc
     3361 attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg acattggtaa
     3421 tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg cagacattac
     3481 gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg cggcggaaga
     3541 agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca agggctccct
     3601 agctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca aagattttgt
     3661 tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg attggttgat
     3721 tatgacaccc ggtgtgggtt tagatgacaa gggagacgca ttgggtcaac agtatagaac
     3781 cgtggatgat gtggtctcta caggatctga cattattatt gttggaagag gactatttgc
     3841 aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct gggaagcata
     3901 tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg catgtatact
     3961 aaactcacaa attagagctt caatttaatt atatcagtta ttacccggga atctcggtcg
     4021 taatgatttc tataatgacg aaaaaaaaaa aattggaaag aaaaagctat tgaaaaagga
     4081 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4141 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4201 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4261 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4321 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4381 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4441 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4501 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4561 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4621 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4681 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4741 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4801 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4861 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4921 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4981 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5041 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5101 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5161 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5221 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5281 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5341 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5401 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5461 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5521 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5581 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5641 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5701 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5761 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     5821 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     5881 cggaagagcg cccaatacgc aaaccgcctc tccccgcg
//
