LOCUS       Exported                6479 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses 6xHis tagged ATG13 [571–700] fragment mutated in F641E, 
            I645E with 14 scramble amino acid sequence DIKLIDTVDLESCN under a Cu
            promoter in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    pCu-His6-Atg13[571–700] F641E, I645E-Ss(424)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6479)
  AUTHORS   Gatica D, Damasio A, Pascual C, Klionsky DJ, Ragusa MJ, Popelka H
  TITLE     The carboxy terminus of yeast Atg13 binds phospholipid membrane via 
            motifs that overlap with the Vac8-interacting domain.
  JOURNAL   Autophagy. 2020 Jun;16(6):1007-1020. doi: 
            10.1080/15548627.2019.1648117. Epub 2019 Aug 2.
  PUBMED    31352862
REFERENCE   2  (bases 1 to 6479)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6479
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      771..1359
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1260..1279
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1513..1530
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1647..1668
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1683..1713
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1721..1737
                     /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     1726..1748
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1745..1761
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1745..1761
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1780..1800
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1782..1800
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1819..2053
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             2077..2094
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2502..2518)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2503..2519)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2521..2768
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2537..2555)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2786..2805)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2787..2805)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2815..2832)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2815..2831)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2824..2846)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2972..3427
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3059..3078)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3269..3290
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3530..4204)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(4205..4486)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4507..4526)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4626..4648
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4686..4704)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4745..6087
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    5077..5124
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        6114..6218
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6219..600
                     /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(6437..6456)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
       61 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
      121 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
      181 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
      241 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
      301 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
      361 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
      421 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
      481 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
      541 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
      601 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
      661 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
      721 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      781 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      841 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      901 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
      961 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1021 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1081 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1141 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1201 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1261 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1321 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1381 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     1441 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     1501 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     1561 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     1621 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     1681 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     1741 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
     1801 gaacaaaagc tggagctcct agttagaaaa agacattttt gctgtcagtc actgtcaaga
     1861 gattcttttg ctggcatttc ttctagaagc aaaaagagcg atgcgtcttt tccgctgaac
     1921 cgttccagca aaaaagacta ccaacgcaat atggattgtc agaatcatat aaaagagaag
     1981 caaataactc cttgtcttgt atcaattgca ttataatatc ttcttgttag tgcaatatca
     2041 tatagaagtc atcgactagt ggatcccctt aagatgcacc atcaccatca tcactctact
     2101 agtgccttga atagtagaag gaattcttta gataagagct caaacaagca gggtatgtca
     2161 ggcttacctc ctatttttgg tggagagagt acttcatatc accacgacaa caaaatacaa
     2221 aagtacaacc aattaggagt agaagaagat gatgatgacg agaatgaccg tttgctcaac
     2281 caaatgggaa acagtgctac aaaagaaaaa agttcagaat ctccaagatc aattgatagc
     2341 atttcaagtt ctttcataaa aagtaggata cctatcagac aaccatacca ttactctcaa
     2401 ccaactactg cgccctttca agctcaggcg aaatttcata aacctgcaaa taagttaatc
     2461 gataatggta ataggagtaa tagttaggat atcaagctta tcgataccgt cgacctcgag
     2521 tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg
     2581 aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt
     2641 tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt
     2701 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt
     2761 taatttgcgg ccggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     2821 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     2881 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     2941 ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg
     3001 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3061 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3121 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3181 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3241 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3301 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3361 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     3421 acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca cacctcatag
     3481 atcggcaagt gcacaaacaa tacttaaata aatactactc agtaataacc tatttcttag
     3541 catttttgac gaaatttgct attttgttag agtcttttac accatttgtc tccacacctc
     3601 cgcttacatc aacaccaata acgccattta atctaagcgc atcaccaaca ttttctggcg
     3661 tcagtccacc agctaacata aaatgtaagc tttcggggct ctcttgcctt ccaacccagt
     3721 cagaaatcga gttccaatcc aaaagttcac ctgtcccacc tgcttctgaa tcaaacaagg
     3781 gaataaacga atgaggtttc tgtgaagctg cactgagtag tatgttgcag tcttttggaa
     3841 atacgagtct tttaataact ggcaaaccga ggaactcttg gtattcttgc cacgactcat
     3901 ctccatgcag ttggacgata tcaatgccgt aatcattgac cagagccaaa acatcctcct
     3961 taggttgatt acgaaacacg ccaaccaagt atttcggagt gcctgaacta tttttatatg
     4021 cttttacaag acttgaaatt ttccttgcaa taaccgggtc aattgttctc tttctattgg
     4081 gcacacatat aatacccagc aagtcagcat cggaatctag agcacattct gcggcctctg
     4141 tgctctgcaa gccgcaaact ttcaccaatg gaccagaact acctgtgaaa ttaataacag
     4201 acatactcca agctgccttt gtgtgcttaa tcacgtatac tcacgtgctc aatagtcacc
     4261 aatgccctcc ctcttggccc tctccttttc ttttttcgac cgaattaatt cttaatcggc
     4321 aaaaaaagaa aagctccgga tcaagattgt acgtaaggtg acaagctatt tttcaataaa
     4381 gaatatcttc cactactgcc atctggcgtc ataactgcaa agtacacata tattacgatg
     4441 ctgttctatt aaatgcttcc tatattatat atatagtaat gtcgttgatc tatggtgcac
     4501 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     4561 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4621 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     4681 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4741 gtatgatcca atatcaaagg aaatgatagc attgaaggat gagactaatc caattgagga
     4801 gtggcagcat atagaacagc taaagggtag tgctgaagga agcatacgat accccgcatg
     4861 gaatgggata atatcacagg aggtactaga ctacctttca tcctacataa atagacgcat
     4921 ataagtacgc atttaagcat aaacacgcac tatgccgttc ttctcatgta tatatatata
     4981 caggcaacac gcagatatag gtgcgacgtg aacagtgagc tgtatgtgcg cagctcgcgt
     5041 tgcattttcg gaagcgctcg ttttcggaaa cgctttgaag ttcctattcc gaagttccta
     5101 ttctctagaa agtataggaa cttcagagcg cttttgaaaa ccaaaagcgc tctgaagacg
     5161 cactttcaaa aaaccaaaaa cgcaccggac tgtaacgagc tactaaaata ttgcgaatac
     5221 cgcttccaca aacattgctc aaaagtatct ctttgctata tatctctgtg ctatatccct
     5281 atataaccta cccatccacc tttcgctcct tgaacttgca tctaaactcg acctctacat
     5341 tttttatgtt tatctctagt attactcttt agacaaaaaa attgtagtaa gaactattca
     5401 tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc tatacgtagt atatagagac
     5461 aaaatagaag aaaccgttca taattttctg accaatgaag aatcatcaac gctatcactt
     5521 tctgttcaca aagtatgcgc aatccacatc ggtatagaat ataatcgggg atgcctttat
     5581 cttgaaaaaa tgcacccgca gcttcgctag taatcagtaa acgcgggaag tggagtcagg
     5641 ctttttttat ggaagagaaa atagacacca aagtagcctt cttctaacct taacggacct
     5701 acagtgcaaa aagttatcaa gagactgcat tatagagcgc acaaaggaga aaaaaagtaa
     5761 tttaagatgc tttgttagaa aaatagcgct ctcgggatgc atttttgtag aacaaaaaag
     5821 aagtatagat tctttgttgg taaaatagcg ctctcgcgtt gcatttctgt tctgtaaaaa
     5881 tgcagctcag attctttgtt tgaaaaatta gcgctctcgc gttgcatttt tgttttacaa
     5941 aaatgaagca cagattcttc gttggtaaaa tagcgctttc gcgttgcatt tctgttctgt
     6001 aaaaatgcag ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc atttttgttc
     6061 tacaaaatga agcacagatg cttcgttcag gtggcacttt tcggggaaat gtgcgcggaa
     6121 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6181 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6241 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6301 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6361 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6421 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagag
//
