LOCUS       sequence-192049-        3313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty BB3 for insertion and overexpression of 4 genes in P. 
            pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3313)
  AUTHORS   Prielhofer R, Barrero JJ, Steuer S, Gassler T, Zahrl R, Baumann K, 
            Sauer M, Mattanovich D, Gasser B, Marx H
  TITLE     GoldenPiCS: a Golden Gate-derived modular cloning system for applied
            synthetic biology in the yeast Pichia pastoris.
  JOURNAL   BMC Syst Biol. 2017 Dec 8;11(1):123. doi: 10.1186/s12918-017-0492-3.
  PUBMED    29221460
REFERENCE   2  (bases 1 to 3313)
  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: "BMC Syst 
            Biol."; date: "2017-12-8"; volume: "11(1):123. doi"; pages: " 
            10.1186/s12918-017-0492-3"
COMMENT     Sequence Label: BB3eH_AE
FEATURES             Location/Qualifiers
     source          1..3313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(62..650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(142..161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     gene            761..2336
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        761..1104
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1105..2133
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1105..2133
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2139..2336
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 cgctgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
       61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      481 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      661 tgatcttttc tacggggtgg agcgcaaaaa accccgcccc tgacagggcg gggttttttc
      721 gcgatcggag accggatccc tcgagggtct ccggagcatg gacatggagg cccagaatac
      781 cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat
      841 ttagcccata catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc
      901 gcgaagcaaa aattacggct cctcgctgca gacgtgcgag cagggaaacg ctcccctcac
      961 agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt
     1021 gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca
     1081 catcacatcc gaacataaac aaccatgggt aaaaagcctg aactcaccgc gacgtctgtc
     1141 gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc
     1201 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     1261 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     1321 ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc
     1381 tcccgccgtg cacagggtgt cacgttgcaa gacctccctg aaaccgaact gcccgctgtt
     1441 ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc
     1501 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     1561 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     1621 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     1681 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     1741 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac
     1801 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     1861 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt
     1921 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     1981 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     2041 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga
     2101 cgccccagca ctcgtccgag ggcaaaggaa taatcagtac tgacaataaa aagattcttg
     2161 ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa
     2221 atgttagcgt gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa
     2281 gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactggctt
     2341 cgctcttaat taagcggccc aaagagtgag aggaaagtac ctgggcaaaa tcacacaatt
     2401 ccaaaccatg ctaaatgaga tttaaagagc aaacgatggc aaaaggcaac cgttataaat
     2461 gtgatctttc ttggcagcta tctgtcaatt tttctaagga acagtgaatt catcatagga
     2521 gagatgttat acgttacata atcatacata ctgcatgtat ctcacctact ttacctcatc
     2581 aactctaaaa cagttctagt cccaacccca gattcctagt catgacacaa gtccgcaccg
     2641 gacaggactc acaaccagca agagaagcta acaaatttac gccccggtaa aacattcttt
     2701 aggggccgtt caatggtaat tttcctctca cccgtttaaa cttacctccg ggcggtatct
     2761 tcaataacct ctgttgtccc cgggtatcat tggaaacagt gagggacgtt gaacagaaga
     2821 gaggatcacc gtaaatttgc cttgcaattg gccctaacca cggatggtta acttcaagcc
     2881 atcacgacag caattgagtc ggcgcatagc taccctcctc ttcttgaccc catgcatagg
     2941 accaacctta accgatggaa caggttcctc cgctccgtcc cctggtagtg tctctgcgca
     3001 agaaatagtt aaggtattaa cactgatctc tcgcaccccc ctcacagtac tgttatggtg
     3061 aattgacaaa gccattggct agattgaaac atgtaattca tatgtaatct tgttcaatta
     3121 acgagcttcg tacagtctca atctagacgt ctgataatgg cgtttgtgct cctaatcgat
     3181 gagccatctc atgtgacgtc tatacgcttc gatggcttcc gtcgcgaata tagaaccact
     3241 tgaaatatgc tgcaaaccac gatccaccct ggtcctgaaa agatataaat acagcacatc
     3301 tagcaggctt ttg
//
