LOCUS       Exported                7140 bp ds-DNA     circular SYN 24-OCT-2017
DEFINITION  Construct for labeling the ER of P. pastoris with an msGFP-HDEL 
            fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7140)
  AUTHORS   Glick Lab
  TITLE     Direct Submission
  JOURNAL   Exported Sep 12, 2018 from SnapGene 4.2.2
            http://www.snapgene.com
COMMENT     Linearize with SalI or StuI for integration at the HIS4 locus.
FEATURES             Location/Qualifiers
     source          1..7140
                     /organism="synthetic DNA construct"
                     /lab_host="Pichia pastoris"
                     /mol_type="other DNA"
     CDS             1..93
                     /codon_start=1
                     /product="signal sequence of P. pastoris Kar2"
                     /label=pre-Kar2
                     /translation="MLSLKPSWLTLAALMYAMLLVVVPFAKPVRA"
     CDS             94..804
                     /codon_start=1
                     /product="monomeric superfolder GFP"
                     /label=msGFP
                     /note="mammalian codon-optimized"
                     /translation="DSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGSSGSSG"
     CDS             814..825
                     /codon_start=1
                     /label=HDEL
                     /translation="HDEL"
     misc_feature    829..1028
                     /label=3' PpKAR2
                     /note="region downstream of the P. pastoris KAR2 coding 
                     sequence"
     rep_origin      complement(1392..1980)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2340..3149)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(3150..3493)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3641..6172
                     /codon_start=1
                     /gene="Pichia pastoris HIS4"
                     /product="multifunctional enzyme, required for histidine 
                     biosynthesis"
                     /label=PpHIS4
                     /note="auxotrophic marker for Pichia pastoris"
                     /translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE
                     LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL
                     VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII
                     DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI
                     PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER
                     TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED
                     IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP
                     KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE
                     LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLAETLQVETCP
                     GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE
                     VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA
                     LCSIDMPAGPSEVLVIADKYADPDFVVSDLLSQAEHGIDSQVILLAVDMTDKELARIED
                     AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASYVDQVQ
                     HAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLKH
                     IGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"
     misc_feature    6173..6212
                     /label=3' UTR
     misc_feature    6818..7140
                     /label=5' PpKAR2
                     /note="region upstream of the P. pastoris KAR2 coding 
                     sequence"
ORIGIN
        1 atgctgtcgt taaaaccatc ttggctgact ttggcggcat taatgtatgc catgctattg
       61 gtcgtagtgc catttgctaa acctgttaga gctgatagca ctgagagcct gttcaccggg
      121 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgcgc
      181 ggcgagggcg agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc
      241 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
      301 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
      361 ggctacgtcc aggagcgcac catcaccttc aaggacgacg gcacctacaa gacccgcgcc
      421 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
      481 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acttcaacag ccacaacgtc
      541 tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac
      601 gtggaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
      661 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccaagct gagcaaagac
      721 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
      781 cacggcagct ccggttccag cgggtcttcc ggtcatgatg agttgtagat aggctaaatt
      841 atatatagga aagaatagta ttacaaatta atggttctaa tttatttcac ctattgaatt
      901 tttttaccac tgagaaacaa tgtttaggta tatgtgctta cgtaatgcca cgaaaattta
      961 tggcatcctt caacttctga aatcaactac tggcggagga agcaaatttt tccaggagca
     1021 acaacagaac tgcaggcatg caagcttctt agacatgact gttcctcagt tcaagttggg
     1081 cacttacgag aagaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg
     1141 cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga
     1201 ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc tatctcgcag
     1261 ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg tttttcttgg
     1321 tatttcccac acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     1381 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     1441 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     1501 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     1561 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     1621 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1681 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1741 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1801 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     1861 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1921 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1981 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2041 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2101 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag tcaatcacat
     2161 acgattgacg catgatatta ctttctgcgc acttaacttc gcatctgggc agatgatgtc
     2221 gaggcgaaaa aaaatataaa tcacgctaac atttgattaa aatagaacaa ctacaatata
     2281 aaaaaactat acaaatgaca agttcttgaa aacaagaatc tttttattgt cagtactgat
     2341 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     2401 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     2461 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     2521 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     2581 gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag
     2641 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     2701 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     2761 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     2821 tcttctaata cctggaatgc tgttttgccg gggatcgcag tggtgagtaa ccatgcatca
     2881 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     2941 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     3001 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     3061 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     3121 ctcgaaacgt gagtcttttc cttacccatg gttgtttatg ttcggatgtg atgtgagaac
     3181 tgtatcctag caagatttta aaaggaagta tatgaaagaa gaacctcagt ggcaaatcct
     3241 aaccttttat atttctctac aggggcgcgg cgtggggaca attcaacgcg tctgtgaggg
     3301 gagcgtttcc ctgctcgcag gtctgcagcg aggagccgta atttttgctt cgcgccgtgc
     3361 ggccatcaaa atgtatggat gcaaatgatt atacatgggg atgtatgggc taaatgtacg
     3421 ggcgacagtc acatcatgcc cctgagctgc gcacgtcaag actgtcaagg agggtattct
     3481 gggcctccat gtcgctggcc gggtgacccg gcggggacga ggcaagctaa acagatctgg
     3541 cgcgccttaa ttaacaatga tctcctgatg actgactcac tgataataaa aatacggctt
     3601 cagaatttct caagactaca ctcactgtcc gacttcaagt atgacatttc ccttgctacc
     3661 tgcatacgca agtgttgcag agtttgataa ttccttgagt ttggtaggaa aagccgtgtt
     3721 tccctatgct gctgaccagc tgcacaacct gatcaagttc actcaatcga ctgagcttca
     3781 agttaatgtg caagttgagt catccgttac agaggaccaa tttgaggagc tgatcgacaa
     3841 cttgctcaag ttgtacaata atggtatcaa tgaagtgatt ttggacctag atttggcaga
     3901 aagagttgtc caaaggatga tcccaggcgc tagggttatc tataggaccc tggttgataa
     3961 agttgcatcc ttgcccgcta atgctagtat cgctgtgcct ttttcttctc cactgggcga
     4021 tttgaaaagt ttcactaatg gcggtagtag aactgtttat gctttttctg agaccgcaaa
     4081 gttggtagat gtgacttcca ctgttgcttc tggtataatc cccattattg atgctcggca
     4141 attgactact gaatacgaac tttctgaaga tgtcaaaaag ttccctgtca gtgaaatttt
     4201 gttggcgtct ttgactactg accgccccga tggtctattc actactttgg tggctgactc
     4261 ttctaattac tcgttgggcc tggtgtactc gtccaaaaag tctattccgg aggctataag
     4321 gacacaaact ggagtctacc aatctcgtcg tcacggtttg tggtataaag gtgctacatc
     4381 tggagcaact caaaagttgc tgggtatcga attggattgt gatggagact gcttgaaatt
     4441 tgtggttgaa caaacaggtg ttggtttctg tcacttggaa cgcacttcct gttttggcca
     4501 atcaaagggt cttagagcca tggaagccac cttgtgggat cgtaagagca atgctccaga
     4561 aggttcttat accaaacggt tatttgacga cgaagttttg ttgaacgcta aaattaggga
     4621 ggaagctgat gaacttgcag aagctaaatc caaggaagat atagcctggg aatgtgctga
     4681 cttattttat tttgcattag ttagatgtgc caagtacggc gtgacgttgg acgaggtgga
     4741 gagaaacctg gatatgaagt ccctaaaggt cactagaagg aaaggagatg ccaagccagg
     4801 atacaccaag gaacaaccta aagaagaatc caaacctaaa gaagtccctt ctgaaggtcg
     4861 tattgaattg tgcaaaattg acgtttctaa ggcctcctca caagaaattg aagatgccct
     4921 tcgtcgtcct atccagaaaa cggaacagat tatggaatta gtcaaaccaa ttgtcgacaa
     4981 tgttcgtcaa aatggtgaca aagccctttt agaactaact gccaagtttg atggagtcgc
     5041 tttgaagaca cctgtgttag aagctccttt cccagaggaa cttatgcaat tgccagataa
     5101 cgttaagaga gccattgatc tctctataga taacgtcagg aaattccatg aagctcaact
     5161 agcggagacg ttgcaagttg agacttgccc tggtgtagtc tgctctcgtt ttgcaagacc
     5221 tattgagaaa gttggcctct atattcctgg tggaaccgca attctgcctt ccacttccct
     5281 gatgctgggt gttcctgcca aagttgctgg ttgcaaagaa attgtttttg catctccacc
     5341 taagaaggat ggcaccctta ccccagaagt catctacgtt gcccacaagg ttggtgctaa
     5401 gtgtatcgtg ctagcaggag gcgcccaggc agtagctgct atggcttacg gaacagaaac
     5461 tgttcctaag tgtgacaaaa tatttggtcc aggaaaccag ttcgttactg ctgccaagat
     5521 gatggttcaa aatgacacat cagccctgtg tagtattgac atgcctgctg ggccttctga
     5581 agttctagtt attgctgata aatacgctga tccagatttc gttgtctcag accttctgtc
     5641 tcaagctgaa catggtattg attcccaggt gattctgttg gctgtcgata tgacagacaa
     5701 ggagcttgcc agaattgaag atgctgttca caaccaagct gtgcagttgc caagggttga
     5761 aattgtacgc aagtgtattg cacactctac aaccctatcg gttgcaacct acgagcaggc
     5821 tttggaaatg tccaatcagt acgctcctga acacttgatc ctgcaaatcg agaatgcttc
     5881 ttatgttgat caagtacaac acgctggatc tgtgtttgtt ggtgcctact ctccagagag
     5941 ttgtggagat tactcctccg gcaccaacca cactttgcca acgtacggat atgcccgtca
     6001 atacagcgga gttaacactg caaccttcca gaagttcatc acttcacaag acgtaactcc
     6061 tgagggactg aaacatattg gccaagcagt gatggatctg gctgctgttg aaggtctaga
     6121 tgctcaccgc aatgctgtta aggttcgtat ggagaaactg ggacttattt aattatttag
     6181 agattttaac ttacatttag attcgataga tcattattga agcatttatc agggttattg
     6241 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6301 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     6361 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     6421 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     6481 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     6541 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     6601 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     6661 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     6721 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     6781 gacggccagt gaattcgagc tcggtacccg gggatccgag ctcctagtga agacttgaga
     6841 tggacatgat aaacaattat agtgaaaata gaaaccataa tacaatattc taatagagga
     6901 accgtttacc tgtggttcct attgtggcct actgttacta gctagtgtaa tacacccttg
     6961 cctcagcttt gcaagttgac aactcagcca aatgatcttt gaatgcgcga aacctcaagg
     7021 tccatcgaat tttctcgaat tttcagtgtt ttcatacagc gtgtcatctt ctttcgcgta
     7081 cttatttaaa tcgtacccag atcccttctt cttccttaat ttcaattcca acactcaaga
//
