LOCUS       sequence-248556-        8993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing integration cassette at IntD1 integration site in
            Yarrowia lipolytica for overexpression of HMG1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8993)
  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 8993)
  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: pCfB8787
FEATURES             Location/Qualifiers
     source          1..8993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    223..256
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             4392..4403
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(6472..6505)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(6712..6731)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(6737..6756)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        6798..6889
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6890..6958,6959..7750)
                     /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     complement(7108..7127)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7872..8460
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8361..8380
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8614..8631
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     8729..8746
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8730..8746
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tattcatcta ttatttatga ttttttgtat atacaatatt tctagtttgt taaagagaat
       61 taagaaaata aatctcgaaa ataataaagg gaaaatcagt ttttgatatc aaaattatac
      121 atgtcaacga taatacaaaa tataatacaa actataagat gttatcagta tttattatgc
      181 atttagaata aattttgtgt cgcccttatt cgactcacta tagaagttcc tattctctag
      241 aaagtatagg aacttcactt cattttattt aaatttgcgg ccgcgaatgc gtgcgatcat
      301 tttcccgtca tatcaaacac atcaccaacg ctcgtaatta gcatcggttc gctaacgtat
      361 tccaaacacg gattcgagca cctcaacaac atgtctcact agcgcgcatg acgcaaaact
      421 tctccaaagc acgggtacat agggcatgtg tctgttagcg tctcgacaag gccgcaatta
      481 cttatgaaac ctcatcggct gtttcagggg gcgagtggct tgcacatttg ctgttaatac
      541 cgagataacg gcgtgtgagg tatagtacaa tgattacatc gaggagcgat cagagagaac
      601 agaacaagtc ggaagatgaa cgcaatcatt ttgacataaa atatcagagc ggaaaaatca
      661 gttgttttta tttatttatt ttttcagaat ggcaccatgg atctacctcg agtctaacta
      721 caccaacata attagtgggt atggtcgttg agtgttgaca aattagtggg aacaatgcag
      781 agtgttagag caatgtcacg tgcaacgctt acgcaactaa catgaatgaa tacgatatac
      841 atcaaagact atgatacgca gtattgcaca ctgtacgagt aagggcacta gccactgcac
      901 tcaagtgaaa ccgttgcccg ggtacgagta tgagtatgta cagtatgttt agtattgtac
      961 ttggacaatg cttgtatcgt acattctcaa gtgtcaaaca taaatatccg ttgctatatc
     1021 ctcgcaccac cacgtagctc gctatatccc tgtgttgaat ccatccatct tggattgcca
     1081 attgtgcaca cagaaccggg cactcacttc cccatccaca cttcaacgga atgcgtgcga
     1141 taacagtgta cgcagtacta tagaggaaca attgccccgg agaagacggc caggccgcct
     1201 agatgacaaa ttcaacaact cacagctgac tttctgccat tgccactagg ggggggcctt
     1261 tttatatggc caagccaagc tctccacgtc ggttgggctg cacccaacaa taaatgggta
     1321 gggttgcacc aacaaaggga tgggatgggg ggtagaagat acgaggataa cggggctcaa
     1381 tggcacaaat aagaacgaat actgccatta agactcgtga tccagcgact gacaccattg
     1441 catcatctaa gggcctcaaa actacctcgg aactgctgcg ctgatctgga caccacagag
     1501 gttccgagca ctttaggttg caccaaatgt cccaccaggt gcaggcagaa aacgctggaa
     1561 cagcgtgtac agtttgtctt agcaaaaagt gaaggcgctg aggtcgagca gggtggtgtg
     1621 acttgttata gcctttagag ctgcgaaagc gcgtatggat ttggctcatc aggccagatt
     1681 gagggtctgt ggacacatgt catgttagtg tacttcaatc gccccctgga tatagccccg
     1741 acaataggcc gtggcctcat ttttttgcct tccgcacatt tccattgctc ggtacccaca
     1801 ccttgcttct cctgcacttg ccaaccttaa tactggttta cattgaccaa catcttacaa
     1861 gcggggggct tgtctagggt atatataaac agtggctctc ccaatcggtt gccagtctct
     1921 tttttccttt ctttccccac agattcgaaa tctaaactac acatcacaca atctgtcatg
     1981 ccacaatgct acaagcagct attggaaaga ttgtgggatt tgcggtcaac cgacccatcc
     2041 acacagttgt cctgacgtcc atcgtggcgt caaccgcata cctcgccatc ctcgacattg
     2101 ccatcccggg tttcgagggc acacaaccca tctcatacta ccaccctgca gcaaaatctt
     2161 acgacaaccc tgctgattgg acccacattg cagaggccga catcccttca gacgcctacc
     2221 gacttgcatt tgcccagatc cgtgtcagtg atgttcaggg cggagaggcc cccaccatcc
     2281 ctggcgccgt ggccgtgtct gatctcgacc acagaatcgt catggactac aaacagtggg
     2341 ccccctggac cgccagcaac gagcagatcg cctcggagaa ccacatctgg aagcactcct
     2401 tcaaggacca cgtggccttc agctggatca agtggttccg atgggcctac ctgcgtttgt
     2461 ccactctcat ccagggggca gacaacttcg acattgccgt ggtcgccctt ggctatcttg
     2521 ccatgcacta caccttcttc agtctcttcc gatccatgcg aaaggttggc tcgcactttt
     2581 ggcttgcctc catggctctg gtctcttcca ccttcgcttt cctgcttgcg gtggtggctt
     2641 cctctagcct gggttaccga cctagcatga tcaccatgtc cgagggcctg cccttcctcg
     2701 tggtcgccat tggctttgac cgaaaggtca acctggctag cgaggtgctc acatccaaga
     2761 gcagccagct cgctcccatg gtgcaggtga tcacaaagat cgcctccaag gcgctgtttg
     2821 agtacagcct tgaggtggcc gccctgtttg ctggcgccta taccggagtt cctcgactgt
     2881 cccagttttg cttcttatct gcttggatcc tcatcttcga ctacatgttt ttgctgacct
     2941 tctactctgc tgtccttgct atcaagtttg agatcaatca cattaagcga aaccgaatga
     3001 tccaggatgc tctcaaggag gatggtgtat ctgctgctgt tgccgagaag gtagccgact
     3061 cttctcccga cgccaagctc gaccgaaagt ccgacgtttc tctttttgga gcctctggcg
     3121 ccattgcggt gttcaagatc ttcatggtcc ttgggttcct tggtctcaac ctcatcaacc
     3181 tgactgccat ccctcacctt ggcaaggcag ccgccgctgc ccagtctgtg actcccatca
     3241 ccctctcccc cgagcttctc catgccatcc ccgcctctgt gcccgttgtt gtcacctttg
     3301 tgcccagcgt tgtgtacgag cactcccagc tcattctgca gctggaggac gccctcacta
     3361 ccttcctggc tgcctgctcc aaaactattg gtgaccccgt catctccaag tacatcttcc
     3421 tgtgcctgat ggtctccacc gccctgaacg tctacctgtt tggagccacc cgagaagttg
     3481 tgcgaaccca gtctgtgaag gtggttgaga agcacgttcc tatcgtcatt gagaagccca
     3541 gcgagaagga ggaggacacc tcttctgaag actccattga gctgactgtc ggaaagcagc
     3601 ccaagcccgt gaccgagacc cgttctctgg acgacctaga ggctatcatg aaggcaggta
     3661 agaccaagct tctggaggac cacgaggttg tcaagctctc tctcgagggc aagcttcctt
     3721 tgtatgctct tgagaagcag cttggtgaca acacccgagc tgttggcatc cgacgatcta
     3781 tcatctccca gcagtctaat accaagactt tagagacctc aaagcttcct tacctgcact
     3841 acgactacga ccgtgttttt ggagcctgtt gcgagaacgt tattggttac atgcctctcc
     3901 ccgttggtgt tgctggcccc atgaacattg atggcaagaa ctaccacatt cctatggcca
     3961 ccactgaggg ttgtcttgtt gcctcaacca tgcgaggttg caaggccatc aacgccggtg
     4021 gcggtgttac cactgtgctt actcaggacg gtatgacacg aggtccttgt gtttccttcc
     4081 cctctctcaa gcgggctgga gccgctaaga tctggcttga ttccgaggag ggtctcaagt
     4141 ccatgcgaaa ggccttcaac tccacctctc gatttgctcg tctccagtct cttcactcta
     4201 cccttgctgg taacctgctg tttattcgat tccgaaccac cactggtgat gccatgggca
     4261 tgaacatgat ctccaagggc gtcgaacact ctctggccgt catggtcaag gagtacggct
     4321 tccctgatat ggacattgtg tctgtctcgg gtaactactg cactgacaag aagcccgcag
     4381 cgatcaactg gatcgaaggc cgaggcaaga gtgttgttgc cgaagccacc atccctgctc
     4441 acattgtcaa gtctgttctc aaaagtgagg ttgacgctct tgttgagctc aacatcagca
     4501 agaatctgat cggtagtgcc atggctggct ctgtgggagg tttcaatgca cacgccgcaa
     4561 acctggtgac cgccatctac cttgccactg gccaggatcc tgctcagaat gtcgagtctt
     4621 ccaactgcat cacgctgatg agcaacgtcg acggtaacct gctcatctcc gtttccatgc
     4681 cttctatcga ggtcggtacc attggtggag gtactatttt ggagccccag ggggctatgc
     4741 tggagatgct tggcgtgcga ggtcctcaca tcgagacccc cggtgccaac gcccaacagc
     4801 ttgctcgcat cattgcttct ggagttcttg cagcggagct ttcgctgtgt tctgctcttg
     4861 ctgccggcca tcttgtgcaa agtcatatga cccacaaccg gtcccaggct cctactccgg
     4921 ccaagcagtc tcaggccgat ctgcagcgtc tacaaaacgg ttcgaatatt tgcatacggt
     4981 catagatcgc gtgcattcct tctgttcgga atcaacctca aggttaacgg ccacgatccc
     5041 ctcgttgtta ctcttggtca gcccattgtc ggtaacgctg gctttgctaa ctgggtcgat
     5101 aaactcttct ttggccagga gaaccccgat gtctccaagg tgtccaaaga ccgaaagctc
     5161 taccgaatca cccaccgagg agatatcgtc cctcaagtgc ccttctggga cggttaccag
     5221 cactgctctg gtgaggtctt tattgactgg cccctgatcc accctcctct ctccaacgtt
     5281 gtcatgtgcc agggccagag caataaacag tgctctgccg gtaacactct gctccagcag
     5341 gtcaatgtga ttggaaacca tctgcagtac ttcgtcaccg agggtgtctg tggtatctaa
     5401 gctatttatc actctttaca acttctacct caactatcta ctttaataaa tgaatatcgt
     5461 ttattctcta tgattactgt atatgcgttc ctctaagaca aatcgaaacc agcatgcgat
     5521 cgaatggcat acaaaagttt cttccgaagt tgatcaatgt cctgatagtc aggcagcttg
     5581 agaagattga cacaggtgga ggccgtaggg aaccgatcaa cctgtctacc agcgttacga
     5641 atggcaaatg acgggttcaa agccttgaat ccttgcaatg gtgccttgga tactgatgtc
     5701 acaaacttaa gaagcagccg cttgtcctct tcctcgaaac tctcaaacac agtccagaag
     5761 tcctttatag tttgatctgt atccagatag cctccgtaat tggtgtgtgt cttcaaatcc
     5821 cagacgtcca cattggcatg tcctccactg ataagcattt gaagttcatc tgcgttgaac
     5881 attgagaccc acgaagggtc aatgagctgg tatagaccgc ccaagaatgc atctgagtgg
     5941 ccttctgcgt ctgctgtttg tgtctttctc gttcgccacg gtccgcctga gtcaccattg
     6001 cccatctact ccccggccca gtcatcgact ttgagcgatg acgccggtta ccggaacctt
     6061 tgcaggggca acgccgacac cccggcgcgt gtggtcgcca ttgcaacggt tcattctagc
     6121 acatgtgcca tgtggccgtg tcgcaccttg tatccacgca gctgctctcc gacatgccca
     6181 aaccctcgcg caatagtgtc aggtttacga gtgtcaatgc gatggcataa ttagtgtttc
     6241 caaccaagcg atattatagc caacatgaaa gcgaggttga cgggcgacga cgcgacggct
     6301 gagttgctga gccttgtgga aacggcggcc attatataac gctcaagaag ctcgaggcgt
     6361 gttttctggc aaccacaccg actcggtggc ccattgcacc ctccacacct ctccactctt
     6421 acccctggcc ggatcgcgtg cattcgcggc cgcatttaaa tccccacttc agaagttcct
     6481 atactttcta gagaatagga acttcactat agagtcgaat aagggcgaca ccccataatt
     6541 agcccgggcg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctggcag
     6601 ttccctactc tcgcatgggg agtccccaca ctaccatcgg cgctacggcg tttcacttct
     6661 gagttcggca tggggtcagg tgggaccacc gcgctactgc cgccaggcaa acaaggggtg
     6721 ttatgagcca tattcaggta taaatgggct cgcgataatg ttcagaattg gttaattggt
     6781 tgtaacactg acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     6841 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagaata tgagtattca
     6901 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     6961 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     7021 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     7081 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     7141 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     7201 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     7261 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     7321 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     7381 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcgat
     7441 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     7501 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     7561 ggctggctgg tttattgctg ataaatccgg agccggtgag cgtggttctc gcggtatcat
     7621 cgcagcgctg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     7681 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     7741 gcattggtaa gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat
     7801 cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc
     7861 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     7921 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     7981 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     8041 gcccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     8101 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     8161 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     8221 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     8281 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     8341 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     8401 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     8461 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     8521 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     8581 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     8641 ggcgagagta gggaactgcc aggcatcaaa ctaagcagaa ggcccctgac ggatggcctt
     8701 tttgcgtttc tacaaactct ttctgtgttg taaaacgacg gccagtctta agctcgggcc
     8761 ccctgggcgg ttctgataac gagtaatcgt taatccgcaa ataacgtaaa aacccgcttc
     8821 ggcgggtttt tttatggggg gagtttaggg aaagagcatt tgtcagaata tttaagggcg
     8881 cctgtcactt tgcttgatat atgagaatta tttaacctta taaatgagaa aaaagcaacg
     8941 cactttaaat aagatacgtt gctttttcga ttgatgaaca cctataatta aac
//
