LOCUS       sequence-248512-        4475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for PCR amplification of pGPD1-pFBA1 double promoter 
            biobrick for Yarrowia lipolytica.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4475)
  AUTHORS   Milne N, Tramontin LRR, Borodina I
  TITLE     A teaching protocol demonstrating the use of EasyClone and 
            CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae 
            and Yarrowia lipolytica.
  JOURNAL   FEMS Yeast Res. 2019 Sep 26. pii: foz062. doi: 
            10.1093/femsyr/foz062.
  PUBMED    31556952
REFERENCE   2  (bases 1 to 4475)
  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: "FEMS Yeast 
            Res. 2019 Sep 26. pii: foz062. doi: 10.1093/femsyr/foz062."
COMMENT     Sequence Label: pCfB8742
FEATURES             Location/Qualifiers
     source          1..4475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..30)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(184..772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(264..283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(943..1734,1735..1803))
                     /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     1566..1585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1804..1908)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1976..1994
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2032..2054)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2154..2173
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2367..2389
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2381..2398
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2382..2398
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4257..4273)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4257..4273)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4270..4292)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4281..4297
                     /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)."
     promoter        complement(join(4305..4311,4312..4329,4330..4335))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4350..4371
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
       61 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1801 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1861 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1921 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     1981 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     2041 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     2101 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     2161 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     2221 gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg
     2281 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     2341 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     2401 attcgagctc ggtacccacc tgcactgttg atgtgtgttt aattcaagaa tgaatataga
     2461 gaagagaaga agaaaaaaga ttcaattgag ccggcgatgc agacccttat ataaatgttg
     2521 ccttggacag acggagcaag cccgcccaaa cctacgttcg gtataatatg ttaagctttt
     2581 taacacaaag gtttggcttg gggtaacctg atgtggtgca aaagaccggg cgttggcgag
     2641 ccattgcgcg ggcgaatggg gccgtgactc gtctcaaatt cgagggcgtg cctcaattcg
     2701 tgcccccgtg gctttttccc gccgtttccg ccccgtttgc accactgcag ccgcttcttt
     2761 ggttcggaca ccttgctgcg agctaggtgc cttgtgctac ttaaaaagtg gcctcccaac
     2821 accaacatga catgagtgcg tgggccaaga cacgttggcg gggtcgcagt cggctcaatg
     2881 gcccggaaaa aacgctgctg gagctggttc ggacgcagtc cgccgcggcg tatcgatatc
     2941 cgcaaggttc catggcgcca ttgccctccg tcggcgtcta tcccgcaacc tctaaataga
     3001 gcgggaatat aacccaagct tctttttttt tcctttaaca cgcacacccc caactatcat
     3061 gttgctgctg ctgtttgact ctactctgtg gaggggtgct cccacccaac ccaacctaca
     3121 ggtggatccg gcgctgtgat tggctgataa gtctcctatc cggactaatt ctgaccaatg
     3181 ggacatgcgc gcaggaccca aatgccgcaa ttacgtaacc ccaacgaaat gcctacccct
     3241 ctttggagcc cagcggcccc aaatcccccc aagcagcccg gttctaccgg cttccatctc
     3301 caagcacccc tttctccaca ccccacaaaa agacccgtgc aggacatcct actgcgtcag
     3361 ctactgataa cagtgtacgc agtactatag aggaacaatt gccccggaga agacggccag
     3421 gccgcctaga tgacaaattc aacaactcac agctgacttt ctgccattgc cactaggggg
     3481 gggccttttt atatggccaa gccaagctct ccacgtcggt tgggctgcac ccaacaataa
     3541 atgggtaggg ttgcaccaac aaagggatgg gatggggggt agaagatacg aggataacgg
     3601 ggctcaatgg cacaaataag aacgaatact gccattaaga ctcgtgatcc agcgactgac
     3661 accattgcat catctaaggg cctcaaaact acctcggaac tgctgcgctg atctggacac
     3721 cacagaggtt ccgagcactt taggttgcac caaatgtccc accaggtgca ggcagaaaac
     3781 gctggaacag cgtgtacagt ttgtcttagc aaaaagtgaa ggcgctgagg tcgagcaggg
     3841 tggtgtgact tgttatagcc tttagagctg cgaaagcgcg tatggatttg gctcatcagg
     3901 ccagattgag ggtctgtgga cacatgtcat gttagtgtac ttcaatcgcc ccctggatat
     3961 agccccgaca ataggccgtg gcctcatttt tttgccttcc gcacatttcc attgctcggt
     4021 acccacacct tgcttctcct gcacttgcca accttaatac tggtttacat tgaccaacat
     4081 cttacaagcg gggggcttgt ctagggtata tataaacagt ggctctccca atcggttgcc
     4141 agtctctttt ttcctttctt tccccacaga ttcgaaatct aaactacaca tcacacaatc
     4201 tgtcatgggg atcctctaga gtcgacctgc aggcatgcaa gcttggcgta atcatggtca
     4261 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4321 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4381 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4441 caacgcgcgg ggagaggcgg tttgcgtatt gggcg
//
