LOCUS       Exported                5784 bp ds-DNA     circular SYN 29-SEP-2022
DEFINITION  Plasmid for the expression and purification of GABARAP. Internal 
            Reference: SMC894.
ACCESSION   .
VERSION     .
KEYWORDS    pET-Duet1_6xHis-TEV-GABARAP-Gly
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5784)
  AUTHORS   Turco E, Savova A, Gere F, Ferrari L, Romanov J, Schuschnig M, 
            Martens S
  TITLE     Reconstitution defines the roles of p62, NBR1 and TAX1BP1 in 
            ubiquitin condensate formation and autophagy initiation.
  JOURNAL   Nat Commun. 2021 Sep 1;12(1):5212. doi: 10.1038/s41467-021-25572-w.
  PUBMED    34471133
REFERENCE   2  (bases 1 to 5784)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 29, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5784
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    3..27
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             113..133
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        581..599
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    600..624
                     /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)."
     CDS             733..777
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(815..833)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      829..876
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      913..1368
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1000..1019)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1210..1231
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1483..2343)
                     /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     2106..2125
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2344..2436)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2517..3105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3006..3025
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3259..3276
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3291..3430
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3356..3375)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3516..3538
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3532..3723)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4295..5377)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5339..5358
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5378..5455)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5661..5680
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5768..2
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cggaaaacct
      121 gtattttcag ggaatgaagt tcgtgtacaa agaagagcat ccgttcgaga agcgccgctc
      181 tgagggcgag aagatccgaa agaaataccc ggaccgggtg ccggtgatag tagaaaaggc
      241 tcccaaagct cggataggag acctggacaa aaagaaatac ctggtgcctt ctgatctcac
      301 agttggtcag ttctacttct tgatccggaa gcgaattcat ctccgagctg aggatgcctt
      361 gtttttcttt gtcaacaatg tcattccacc caccagtgcc acaatgggtc agctgtacca
      421 ggaacaccat gaagaagact tctttctcta cattgcctac agtgacgaaa gtgtctacgg
      481 ttgagagctc ggcgcgcctg caggtcgaca agcttgcggc cgcataatgc ttaagtcgaa
      541 cagaaagtaa tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg
      601 gaattgtgag cggataacaa ttccccatct tagtatatta gttaagtata agaaggagat
      661 atacatatgg cagatctcaa ttggatatcg gccggccacg cgatcgctga cgtcggtacc
      721 ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct
      781 actagcgcag cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc
      841 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
      901 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      961 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1021 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1081 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1141 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1201 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1261 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1321 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttct ggcggcacga
     1381 tggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     1441 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     1501 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1561 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1621 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1681 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1741 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1801 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1861 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1921 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1981 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     2041 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     2101 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     2161 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     2221 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     2281 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     2341 catactcttc ctttttcaat catgattgaa gcatttatca gggttattgt ctcatgagcg
     2401 gatacatatt tgaatgtatt tagaaaaata aacaaatagg tcatgaccaa aatcccttaa
     2461 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2521 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2581 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2641 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2701 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2761 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2821 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2881 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2941 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3001 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3061 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3121 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3181 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3241 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3301 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3361 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3421 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3481 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3541 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     3601 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     3661 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     3721 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     3781 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     3841 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     3901 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     3961 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4021 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4081 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4141 ccagcctagc cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca
     4201 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     4261 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4321 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4381 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     4441 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     4501 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     4561 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     4621 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     4681 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     4741 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4801 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4861 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4921 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4981 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5041 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5101 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     5161 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     5221 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     5281 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     5341 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     5401 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     5461 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     5521 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     5581 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     5641 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     5701 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc
     5761 gcgaaattaa tacgactcac tata
//
