LOCUS       sequence-316107-        6695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-Vrg4 in yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6695)
  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 6695)
  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-sGFP-Vrg4
FEATURES             Location/Qualifiers
     source          1..6695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    119..140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(155..160,161..178,179..185)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    193..209
                     /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     198..220
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     217..233
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     217..233
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             739..1452
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MGKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(769..797)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1392..1412
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3206..3223)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3206..3222)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3215..3237)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3431..3450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3550..3572
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3690..4493)
                     /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(4494..4709)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4716..4734)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4802..4906
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4907..4975,4976..5767)
                     /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(5125..5144)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5938..6526
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6427..6446
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(6680..6695,1..2)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
       61 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
      121 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      181 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
      241 acgccaagct tgtaatggac catggcaatg tgcgccatat atatatgcgg ctcccttatg
      301 agtagaagaa aatatatgac tggtggtaca aagtggcatg gtggtatata gatgtaggaa
      361 caccatttga gtttgccata atgattaaat ttcccttttt ataaagaatt agccgcccat
      421 aacacgaaaa atatcacagt tgacgaaaga agacacgtcg cctaaacgcg taatgaatcg
      481 caatatacga aaagctaaac gggtttttca gaatcctctt acccagcctt ggataatagt
      541 tttggccgaa aaaaaaaaaa agaaagaaaa aaattgaaat ttcgcgatcc gaacaaacaa
      601 tgaacagaaa aacagaatat atataacgct gccaagccta tttcttgttg ccgattaatt
      661 gttgacatcc tcttaattac caaaagagcc taagaaaaca aacacactaa ccacacagta
      721 tctttcgccc gaatgagcat gggtaaagga gaagaacttt tcactggagt tgtcccaatt
      781 cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa
      841 ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct
      901 gttccttggc caacacttgt cactactttc acttatggtg ttcagtgctt ttcaagatac
      961 ccggatcata tgaaacggca tgactttttc aagagtgcca tgcccgaagg ttatgtacag
     1021 gaaagaacta tatttttcaa agatgacggg aactacaaga cacgtgctga agtcaagttt
     1081 gaaggtgata cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga
     1141 aacattcttg ggcacaaatt ggaatacaac tataactcac acaatgtata catcatggca
     1201 gacaaacaaa agaatggaat caaagctaac ttcaaaatta gacacaacat tgaagatgga
     1261 agcgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt
     1321 ttaccagaca accattacct gtccacacaa tctgcccttt cgaaagatcc caacgaaaag
     1381 agagaccaca tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat
     1441 gaactataca aatcatgctc agaattgaaa acaggtcatg caggccataa cccttgggct
     1501 tcagttgcca attccggtcc gatctctatt ttatcctact gtggttcctc tattttaatg
     1561 acggtgacta acaagttcgt cgtcaatttg aaggatttca acatgaactt tgtcatgctt
     1621 ttcgtgcaat ctttggtttg tactataacc ttgattatcc tacgtatact gggctatgcg
     1681 aagttccgtt cattaaacaa aacagacgcc aagaactggt tccctatttc ctttttactg
     1741 gtcttgatga tctacacctc ttcgaaggct ttacaatact tggctgttcc aatttacacc
     1801 attttcaaga atttgactat tatcttgatt gcttatggtg aggttctctt ttttggtggc
     1861 tctgtcacct ccatggaatt gtcatcattt ttgttgatgg tcctttcttc tgtcgttgca
     1921 acttggggtg accagcaagc tgtggctgcc aaggctgctt cattggctga aggagcagcc
     1981 ggtgctgttg cctcctttaa cccaggttat ttctggatgt tcaccaactg tatcacttct
     2041 gcattattcg ttcttataat gagaaagaga attaagttaa ctaacttcaa ggatttcgac
     2101 actatgtttt acaacaatgt tttggctcta cctattctat tgctgttttc tttctgtgtg
     2161 gaagattggt cttcagttaa tttgaccaat aacttttcta acgattcgct aactgctatg
     2221 atcatcagtg gtgttgcatc cgtcggtatt tcttactgtt ccggttggtg tgttcgtgtt
     2281 acttcgtcta ctacatattc gatggtaggg gctttgaaca agctgccaat tgccttgtct
     2341 ggtttgattt tctttgatgc tccaagaaac ttcttatcta ttctctccat ttttattggt
     2401 ttcctatcag gtattattta tgctgttgcc aaacaaaaga agcaacaagc ccaaccttta
     2461 cgtaaatgag aacttacggg gggtgcaatt tatttttttt ttttggttta ttttatttta
     2521 tagaggcatc taatgcaagt agatttatat acaattatac ttaaaattga tataccttag
     2581 aacaggtcgg acacaacgtg tggtaactcc aatatatcac taatgacaat agcgccttct
     2641 ggtgtttgcc ccaaaatttc atttacttca ttctcaacta aatgaataca tgttttcatg
     2701 cccaatttga tacctgtctc gatgtttttc ccactgtcat ctataaaata agcattctcg
     2761 tatcttgcca acccgctttc cttcatggct ttttcaaatg ctttaacatg tggtttgcag
     2821 accagcgtat ctgtcctaga gtagtcacaa tatgtcaatc catcaaataa atccgctata
     2881 cccaacaatc tcaggcaacg tattgcgtgg tttttataag cgttggtgaa aagccacaat
     2941 ttatcaatct ttcccgattg tctcaatctc aacagcatat tccttaatgg tatatcaggt
     3001 ttcaagatat cttgtagtgg taaggaatca tccacaagcc ggttatattc cagtgcgttc
     3061 actttgtgga acattacaag tccccttatg gcaagtccat actctttgta gtaagagtta
     3121 ttcaaaacgt gagcatcttc aggtgatagt ttcaagtgtg tttgaaagaa cctcagtata
     3181 gattgttgca taagatcgtg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     3241 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     3301 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     3361 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     3421 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3481 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3541 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3601 gacgaaaggg cgggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt
     3661 atacatgcat ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat
     3721 gcttcccagc ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca
     3781 aatagtcctc ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt
     3841 ctatactgtt gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc
     3901 aaccaatcgt aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca
     3961 aaatctttgt cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg
     4021 gagcccttgc atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct
     4081 tctgccgcct gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta
     4141 atgtctgccc attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta
     4201 ccaatgtcag caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc
     4261 tttagcggct taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt
     4321 agtaaacaaa ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga
     4381 acatccaatg aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca
     4441 gcaacaggac taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat
     4501 cttcgtttcc tgcatgtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat
     4561 gtttcttcaa cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt
     4621 tcttccttct gttcggagat taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa
     4681 aaagaataaa aaaaaaatga tgaattgaaa agctggcctc gtgatacgcc tatttttata
     4741 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4801 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4861 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4921 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4981 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5041 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5101 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5161 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5221 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5281 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5341 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5401 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5461 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5521 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5581 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5641 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5701 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5761 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5821 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5881 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5941 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6001 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6061 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6121 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6181 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6241 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6301 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6361 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6421 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6481 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6541 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6601 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     6661 cagccgaacg accgagcgca gcgagtcagt gagcg
//
