LOCUS       Exported                7943 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expressing MCP-2yeGFP.
ACCESSION   .
VERSION     .
KEYWORDS    pCu-MCP-2yeGFP(405)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7943)
  TITLE     The Pat1-Lsm Complex Stabilizes ATG mRNA during Nitrogen 
            Starvation-Induced Autophagy
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7943)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7943
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     59..81
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     73..90
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     74..90
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     100..119
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        100..118
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     143..159
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     144..160
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        193..427
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             438..824
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2
                     /note="binds to a specific stem-loop structure in the viral
                     RNA (Peabody, 1993)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             876..1589
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1641..2354
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2364..2381
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2415..2662
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2431..2449)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2694..2714)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2694..2712)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2733..2749)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2733..2749)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2746..2768)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2757..2773
                     /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)."
     promoter        complement(2781..2811)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2826..2847
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2964..2981)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3135..3723)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3215..3234)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3894..4754)
                     /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     4517..4536
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4755..4859)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4927..4945
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4983..5005)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5105..5124
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(5618..6712)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(6713..7120)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(7421..7876)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7558..7579)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7770..7789
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
       61 cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc
      121 gaattgggta ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgaattc
      181 ctgcagcccg ggctagttag aaaaagacat ttttgctgtc agtcactgtc aagagattct
      241 tttgctggca tttcttctag aagcaaaaag agcgatgcgt cttttccgct gaaccgttcc
      301 agcaaaaaag actaccaacg caatatggat tgtcagaatc atataaaaga gaagcaaata
      361 actccttgtc ttgtatcaat tgcattataa tatcttcttg ttagtgcaat atcatataga
      421 agtcatcgac tagtatggct tctaacttta ctcagttcgt tctcgtcgac aatggcggaa
      481 ctggcgacgt gactgtcgcc ccaagcaact tcgctaacgg ggtcgctgaa tggatcagct
      541 ctaactcgcg ttcacaggct tacaaagtaa cctgtagcgt tcgtcagagc tctgcgcaga
      601 atcgcaaata caccatcaaa gtcgaggtgc ctaaagtggc aacccagact gttggtggtg
      661 tagagcttcc tgtagccgca tggcgttcgt acctaaatat ggaactaacc attccaattt
      721 tcgccacgaa ttccgactgc gagcttattg ttaaggcaat gcaaggtctc ctaaaagatg
      781 gaaacccgat tccctcagca atcgcagcaa actccggcat ctacggatcc cccgggctgc
      841 aggaattcga tatcaagctt atcgataccg tcgacatgtc taaaggtgaa gaattattca
      901 ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac aaattttctg
      961 tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa tttatttgta
     1021 ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaact tatggtgttc
     1081 aatgtttttc tagataccca gatcatatga aacaacatga ctttttcaag tctgccatgc
     1141 cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac tacaagacca
     1201 gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta aaaggtattg
     1261 attttaaaga agatggtaac attttaggtc acaaattgga atacaactat aactctcaca
     1321 atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc aaaattagac
     1381 acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat actccaattg
     1441 gtgatggtcc agtcttgtta ccagacaacc attacttatc cactcaatct gccttatcca
     1501 aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact gctgctggta
     1561 ttacccatgg tatggatgaa ttgtacaaaa gatcccccgg gctgcaggaa ttcgatatca
     1621 agcttatcga taccgtcgac atgtctaaag gtgaagaatt attcactggt gttgtcccaa
     1681 ttttggttga attagatggt gatgttaatg gtcacaaatt ttctgtctcc ggtgaaggtg
     1741 aaggtgatgc tacttacggt aaattgacct taaaatttat ttgtactact ggtaaattgc
     1801 cagttccatg gccaacctta gtcactactt taacttatgg tgttcaatgt ttttctagat
     1861 acccagatca tatgaaacaa catgactttt tcaagtctgc catgccagaa ggttatgttc
     1921 aagaaagaac tatttttttc aaagatgacg gtaactacaa gaccagagct gaagtcaagt
     1981 ttgaaggtga taccttagtt aatagaatcg aattaaaagg tattgatttt aaagaagatg
     2041 gtaacatttt aggtcacaaa ttggaataca actataactc tcacaatgtt tacatcatgg
     2101 ctgacaaaca aaagaatggt atcaaagtta acttcaaaat tagacacaac attgaagatg
     2161 gttctgttca attagctgac cattatcaac aaaatactcc aattggtgat ggtccagtct
     2221 tgttaccaga caaccattac ttatccactc aatctgcctt atccaaagat ccaaacgaaa
     2281 agagagacca catggtcttg ttagaatttg ttactgctgc tggtattacc catggtatgg
     2341 atgaattgta caaataaacc ggtcatcatc accatcacca ttgagtttaa acccgctgat
     2401 cctagagggc cgcatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac
     2461 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt
     2521 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct
     2581 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
     2641 acgctcgaag gctttaattt gcaagctccg cggtggagct ccagcttttg ttccctttag
     2701 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     2761 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     2821 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2881 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2941 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3001 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3061 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3121 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3181 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3241 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3301 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3361 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3421 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3481 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3541 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     3601 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3661 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3721 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3781 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3841 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3901 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3961 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4021 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4081 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4141 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4201 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4261 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4321 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4381 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4441 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4501 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4561 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4621 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4681 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4741 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4801 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4861 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     4921 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     4981 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     5041 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     5101 tatgcggcat cagagcagat tgtactgaga gtgcaccata tcgactacgt cgttaaggcc
     5161 gtttctgaca gagtaaaatt cttgagggaa ctttcaccat tatgggaaat ggttcaagaa
     5221 ggtattgact taaactccat caaatggtca ggtcattgag tgttttttat ttgttgtatt
     5281 tttttttttt tagagaaaat cctccaatat ataaattagg aatcatagtt tcatgatttt
     5341 ctgttacacc taactttttg tgtggtgccc tcctccttgt caatattaat gttaaagtgc
     5401 aattcttttt ccttatcacg ttgagccatt agtatcaatt tgcttacctg tattccttta
     5461 catcctcctt tttctccttc ttgataaatg tatgtagatt gcgtatatag tttcgtctac
     5521 cctatgaaca tattccattt tgtaatttcg tgtcgtttct attatgaatt tcatttataa
     5581 agtttatgta caaatatcat aaaaaaagag aatcttttta agcaaggatt ttcttaactt
     5641 cttcggcgac agcatcaccg acttcggtgg tactgttgga accacctaaa tcaccagttc
     5701 tgatacctgc atccaaaacc tttttaactg catcttcaat ggccttacct tcttcaggca
     5761 agttcaatga caatttcaac atcattgcag cagacaagat agtggcgata gggttgacct
     5821 tattctttgg caaatctgga gcagaaccgt ggcatggttc gtacaaacca aatgcggtgt
     5881 tcttgtctgg caaagaggcc aaggacgcag atggcaacaa acccaaggaa cctgggataa
     5941 cggaggcttc atcggagatg atatcaccaa acatgttgct ggtgattata ataccattta
     6001 ggtgggttgg gttcttaact aggatcatgg cggcagaatc aatcaattga tgttgaacct
     6061 tcaatgtagg gaattcgttc ttgatggttt cctccacagt ttttctccat aatcttgaag
     6121 aggccaaaac attagcttta tccaaggacc aaataggcaa tggtggctca tgttgtaggg
     6181 ccatgaaagc ggccattctt gtgattcttt gcacttctgg aacggtgtat tgttcactat
     6241 cccaagcgac accatcacca tcgtcttcct ttctcttacc aaagtaaata cctcccacta
     6301 attctctgac aacaacgaag tcagtacctt tagcaaattg tggcttgatt ggagataagt
     6361 ctaaaagaga gtcggatgca aagttacatg gtcttaagtt ggcgtacaat tgaagttctt
     6421 tacggatttt tagtaaacct tgttcaggtc taacactacc ggtaccccat ttaggaccac
     6481 ccacagcacc taacaaaacg gcatcagcct tcttggaggc ttccagcgcc tcatctggaa
     6541 gtggaacacc tgtagcatcg atagcagcac caccaattaa atgattttcg aaatcgaact
     6601 tgacattgga acgaacatca gaaatagctt taagaacctt aatggcttcg gctgtgattt
     6661 cttgaccaac gtggtcacct ggcaaaacga cgatcttctt aggggcagac attagaatgg
     6721 tatatccttg aaatatatat atatatattg ctgaaatgta aaaggtaaga aaagttagaa
     6781 agtaagacga ttgctaacca cctattggaa aaaacaatag gtccttaaat aatattgtca
     6841 acttcaagta ttgtgatgca agcatttagt catgaacgct tctctattct atatgaaaag
     6901 ccggttccgg cgctctcacc tttccttttt ctcccaattt ttcagttgaa aaaggtatat
     6961 gcgtcaggcg acctctgaaa ttaacaaaaa atttccagtc atcgaatttg attctgtgcg
     7021 atagcgcccc tgtgtgttct cgttatgttg aggaaaaaaa taatggttgc taagagattc
     7081 gaactcttgc atcttacgat acctgagtat tcccacagtt aactgcggtc aagatatttc
     7141 ttgaatcagg cgccttagac cgctcggcca aacaaccaat tacttgttga gaaatagagt
     7201 ataattatcc tataaatata acgtttttga acacacatga acaaggaagt acaggacaat
     7261 tgattttgaa gagaatgtgg attttgatgt aattgttggg attccatttt taataaggca
     7321 ataatattag gtatgtagat atactagaag ttctcctcga ccggtcgata tgcggtgtga
     7381 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt
     7441 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     7501 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     7561 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     7621 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     7681 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     7741 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     7801 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     7861 ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     7921 tgcgggcctc ttcgctatta cgc
//
