LOCUS       sequence-231132-        7143 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Localisation of Endoplasmic Reticulum.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7143)
  AUTHORS   Barrero JJ, Casler JC, Valero F, Ferrer P, Glick BS
  TITLE     An improved secretion signal enhances the secretion of model 
            proteins from Pichia pastoris.
  JOURNAL   Microb Cell Fact. 2018 Oct 12;17(1):161. doi: 
            10.1186/s12934-018-1009-5.
  PUBMED    30314480
REFERENCE   2  (bases 1 to 7143)
  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"; journalName: "Microb Cell
            Fact."; date: "2018-10-12"; volume: "17(1):161. doi"; pages: " 
            10.1186/s12934-018-1009-5"
COMMENT     Sequence Label: pKar2-msGFP-HDEL
FEATURES             Location/Qualifiers
     source          1..7143
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(133..154)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     741..762
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      complement(1392..1980)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1472..1491)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     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"
     primer_bind     3063..3082
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3150..3493)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3641..6175
                     /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
                     VDKVASLPANASIAVPFSSLLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII
                     DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI
                     PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER
                     TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED
                     IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP
                     KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE
                     LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVETCP
                     GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE
                     VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA
                     LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED
                     AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV
                     QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYAHQYSGVNTATFQKFITSQDVTPEGLK
                     HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"
     primer_bind     6371..6389
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6427..6449)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6549..6568
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6762..6784
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6776..6793
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6777..6793
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
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 ttctcatagc 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 agaacagtat 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 gcatcggggc 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 tactgggcga
     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 caagtacggt 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 aacggagacg 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 gttgcctcag 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 ttcttatgtt gatcaagtac aacacgctgg atctgtgttt gttggtgcct actctccaga
     5941 gagttgtgga gattactcct ccggcaccaa ccacactttg ccaacgtacg gatatgccca
     6001 tcaatacagc ggagttaaca ctgcaacctt ccagaagttc atcacttcac aagacgtaac
     6061 tcctgaggga ctgaaacata ttggccaagc agtgatggat ctggctgctg ttgaaggtct
     6121 agatgctcac cgcaatgctg ttaaggttcg tatggagaaa ctgggactta tttaattatt
     6181 tagagatttt aacttacatt tagattcgat agatcattat tgaagcattt atcagggtca
     6241 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6301 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     6361 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     6421 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     6481 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     6541 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     6601 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa
     6661 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     6721 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     6781 aacgacggcc agtgaattcg agctcggtac ccggggatcc gagctcctag tgaagacttg
     6841 agatggacat gataaacaat tatagtgaaa atagaaacca taatacaata ttctaataga
     6901 ggaaccgttt acctgtggtt cctattgtgg cctactgtta ctagctagtg taatacaccc
     6961 ttgcctcagc tttgcaagtt gacaactcag ccaaatgatc tttgaatgcg cgaaacctca
     7021 aggtccatcg aattttctcg aattttcagt gttttcatac agcgtgtcat cttctttcgc
     7081 gtacttattt aaatcgtacc cagatccctt cttcttcctt aatttcaatt ccaacactca
     7141 aga
//
