LOCUS       sequence-311564-        6700 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 6700)
  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 6700)
  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..6700
                     /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             726..1439
                     /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(756..784)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1379..1399
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3193..3210)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3193..3209)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3202..3224)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3418..3437)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3537..3559
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3677..4480)
                     /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(4481..4701)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4708..4726)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4794..4898
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4899..4967,4968..5759)
                     /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(5117..5136)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5930..6518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6419..6438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6672..6689
                     /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 ccaagcttgt
      241 aatggaccat ggcaatgtgc gccatatata tatgcggctc ccttatgagt agaagaaaat
      301 atatgactgg tggtacaaag tggcatggtg gtatatagat gtaggaacac catttgagtt
      361 tgccataatg attaaatttc cctttttata aagaattagc cgcccataac acgaaaaata
      421 tcacagttga cgaaagaaga cacgtcgcct aaacgcgtaa tgaatcgcaa tatacgaaaa
      481 gctaaacggg tttttcagaa tcctcttacc cagccttgga taatagtttt ggccgaaaaa
      541 aaaaaaaaga aagaaaaaaa ttgaaatttc gcgatccgaa caaacaatga acagaaaaac
      601 agaatatata taacgctgcc aagcctattt cttgttgccg attaattgtt gacatcctct
      661 taattaccaa aagagcctaa gaaaacaaac acactaacca cacagtatct ttcgcccgaa
      721 tgagcatggg taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag
      781 atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat
      841 acggaaaact tacccttaaa tttatttgca ctactggaaa actacctgtt ccttggccaa
      901 cacttgtcac tactttcact tatggtgttc agtgcttttc aagatacccg gatcatatga
      961 aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa agaactatat
     1021 ttttcaaaga tgacgggaac tacaagacac gtgctgaagt caagtttgaa ggtgataccc
     1081 ttgttaatag aatcgagtta aaaggtattg attttaaaga agatggaaac attcttggac
     1141 acaaattgga atacaactat aactcacaca atgtatacat catggcagac aaacaaaaga
     1201 atggaatcaa agctaacttc aaaattagac acaacattga agatggaagc gttcaactag
     1261 cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc
     1321 attacctgtc cacacaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg
     1381 tccttcttga gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaat
     1441 catgctcaga attgaaaaca ggtcatgcag gccataaccc ttgggcttca gttgccaatt
     1501 ccggtccgat ctctatttta tcctactgtg gttcctctat tttaatgacg gtgactaaca
     1561 agttcgtcgt caatttgaag gatttcaaca tgaactttgt catgcttttc gtgcaatctt
     1621 tggtttgtac tataaccttg attatcctac gtatactggg ctatgcgaag ttccgttcat
     1681 taaacaaaac agacgccaag aactggttcc ctatttcctt tttactggtc ttgatgatct
     1741 acacctcttc gaaggcttta caatacttgg ctgttccaat ttacaccatt ttcaagaatt
     1801 tgactattat cttgattgct tatggtgagg ttctcttttt tggtggctct gtcacctcca
     1861 tggaattgtc atcatttttg ttgatggtcc tttcttctgt cgttgcaact tggggtgacc
     1921 agcaagctgt ggctgccaag gctgcttcat tggctgaagg agcagccggt gctgttgcct
     1981 cctttaaccc aggttatttc tggatgttca ccaactgtat cacttctgca ttattcgttc
     2041 ttataatgag aaagagaatt aagttaacta acttcaagga tttcgacact atgttttaca
     2101 acaatgtttt ggctctacct attctattgc tgttttcttt ctgtgtggaa gattggtctt
     2161 cagttaattt gaccaataac ttttctaacg attcgctaac tgctatgatc atcagtggtg
     2221 ttgcatccgt cggtatttct tactgttccg gttggtgtgt tcgtgttact tcgtctacta
     2281 catattcgat ggtaggggct ttgaacaagc tgccaattgc cttgtctggt ttgattttct
     2341 ttgatgctcc aagaaacttc ttatctattc tctccatttt tattggtttc ctatcaggta
     2401 ttatttatgc tgttgccaaa caaaagaagc aacaagccca acctttacgt aaatgagaac
     2461 ttacgggggg tgcaatttat tttttttttt tggtttattt tattttatag aggcatctaa
     2521 tgcaagtaga tttatataca attatactta aaattgatat accttagaac aggtcggaca
     2581 caacgtgtgg taactccaat atatcactaa tgacaatagc gccttctggt gtttgcccca
     2641 aaatttcatt tacttcattc tcaactaaat gaatacatgt tttcatgccc aatttgatac
     2701 ctgtctcgat gtttttccca ctgtcatcta taaaataagc attctcgtat cttgccaacc
     2761 cgctttcctt catggctttt tcaaatgctt taacatgtgg tttgcagacc agcgtatctg
     2821 tcctagagta gtcacaatat gtcaatccat caaataaatc cgctataccc aacaatctca
     2881 ggcaacgtat tgcgtggttt ttataagcgt tggtgaaaag ccacaattta tcaatctttc
     2941 ccgattgtct caatctcaac agcatattcc ttaatggtat atcaggtttc aagatatctt
     3001 gtagtggtaa ggaatcatcc acaagccggt tatattccag tgcgttcact ttgtggaaca
     3061 ttacaagtcc ccttatggca agtccatact ctttgtagta agagttattc aaaacgtgag
     3121 catcttcagg tgatagtttc aagtgtgttt gaaagaacct cagtatagat tgttgcataa
     3181 gatcgtgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     3241 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     3301 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     3361 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac
     3421 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     3481 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3541 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcgg
     3601 gtaataactg atataattaa attgaagctc taatttgtga gtttagtata catgcattta
     3661 cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct tcccagcctg
     3721 cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat agtcctcttc
     3781 caacaataat aatgtcagat cctgtagaga ccacatcatc cacggttcta tactgttgac
     3841 ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac caatcgtaac
     3901 cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa tctttgtcgc
     3961 tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag cccttgcatg
     4021 acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct gccgcctgct
     4081 tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg tctgcccatt
     4141 ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca atgtcagcaa
     4201 attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt agcggcttaa
     4261 ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt aaacaaattt
     4321 tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca tccaatgaag
     4381 cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca acaggactag
     4441 gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt cgtttcctgc
     4501 atgtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt tcttcaacac
     4561 tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct tccttctgtt
     4621 cggagattac cgaatcaaaa aaatttcaaa gaaaccgaaa tcaaaaaaaa gaataaaaaa
     4681 aaaatgatga attgaattga aaagctggcc tcgtgatacg cctattttta taggttaatg
     4741 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4801 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4861 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4921 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4981 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5041 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5101 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5161 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5221 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5281 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5341 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5401 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5461 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5521 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5581 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     5641 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5701 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5761 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5821 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5881 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5941 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6001 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6061 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6121 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6181 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6241 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6301 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6361 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6421 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6481 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6541 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     6601 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     6661 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
//
