LOCUS       sequence-248789-        5709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for PCR amplification of HMG1from Yarrowia lipolytica.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5709)
  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 5709)
  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: pCfB8818
FEATURES             Location/Qualifiers
     source          1..5709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     173..191
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(229..251)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     351..370
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     564..586
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     578..595
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     579..595
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3036..3047
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3688..3704)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3688..3704)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3701..3723)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3712..3728
                     /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(3736..3742,3743..3760,3761..3766))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3781..3802
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3919..3936)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4090..4678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4170..4189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4849..5640,5641..5709))
                     /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     5472..5491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
      181 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
      241 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      301 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      361 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
      421 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
      481 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
      541 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
      601 cgagctcggt acccatctgt catgccacaa tgctacaagc agctattgga aagattgtgg
      661 gatttgcggt caaccgaccc atccacacag ttgtcctgac gtccatcgtg gcgtcaaccg
      721 catacctcgc catcctcgac attgccatcc cgggtttcga gggcacacaa cccatctcat
      781 actaccaccc tgcagcaaaa tcttacgaca accctgctga ttggacccac attgcagagg
      841 ccgacatccc ttcagacgcc taccgacttg catttgccca gatccgtgtc agtgatgttc
      901 agggcggaga ggcccccacc atccctggcg ccgtggccgt gtctgatctc gaccacagaa
      961 tcgtcatgga ctacaaacag tgggccccct ggaccgccag caacgagcag atcgcctcgg
     1021 agaaccacat ctggaagcac tccttcaagg accacgtggc cttcagctgg atcaagtggt
     1081 tccgatgggc ctacctgcgt ttgtccactc tcatccaggg ggcagacaac ttcgacattg
     1141 ccgtggtcgc ccttggctat cttgccatgc actacacctt cttcagtctc ttccgatcca
     1201 tgcgaaaggt tggctcgcac ttttggcttg cctccatggc tctggtctct tccaccttcg
     1261 ctttcctgct tgcggtggtg gcttcctcta gcctgggtta ccgacctagc atgatcacca
     1321 tgtccgaggg cctgcccttc ctcgtggtcg ccattggctt tgaccgaaag gtcaacctgg
     1381 ctagcgaggt gctcacatcc aagagcagcc agctcgctcc catggtgcag gtgatcacaa
     1441 agatcgcctc caaggcgctg tttgagtaca gccttgaggt ggccgccctg tttgctggcg
     1501 cctataccgg agttcctcga ctgtcccagt tttgcttctt atctgcttgg atcctcatct
     1561 tcgactacat gtttttgctg accttctact ctgctgtcct tgctatcaag tttgagatca
     1621 atcacattaa gcgaaaccga atgatccagg atgctctcaa ggaggatggt gtatctgctg
     1681 ctgttgccga gaaggtagcc gactcttctc ccgacgccaa gctcgaccga aagtccgacg
     1741 tttctctttt tggagcctct ggcgccattg cggtgttcaa gatcttcatg gtccttgggt
     1801 tccttggtct caacctcatc aacctgactg ccatccctca ccttggcaag gcagccgccg
     1861 ctgcccagtc tgtgactccc atcaccctct cccccgagct tctccatgcc atccccgcct
     1921 ctgtgcccgt tgttgtcacc tttgtgccca gcgttgtgta cgagcactcc cagctcattc
     1981 tgcagctgga ggacgccctc actaccttcc tggctgcctg ctccaaaact attggtgacc
     2041 ccgtcatctc caagtacatc ttcctgtgcc tgatggtctc caccgccctg aacgtctacc
     2101 tgtttggagc cacccgagaa gttgtgcgaa cccagtctgt gaaggtggtt gagaagcacg
     2161 ttcctatcgt cattgagaag cccagcgaga aggaggagga cacctcttct gaagactcca
     2221 ttgagctgac tgtcggaaag cagcccaagc ccgtgaccga gacccgttct ctggacgacc
     2281 tagaggctat catgaaggca ggtaagacca agcttctgga ggaccacgag gttgtcaagc
     2341 tctctctcga gggcaagctt cctttgtatg ctcttgagaa gcagcttggt gacaacaccc
     2401 gagctgttgg catccgacga tctatcatct cccagcagtc taataccaag actttagaga
     2461 cctcaaagct tccttacctg cactacgact acgaccgtgt ttttggagcc tgttgcgaga
     2521 acgttattgg ttacatgcct ctccccgttg gtgttgctgg ccccatgaac attgatggca
     2581 agaactacca cattcctatg gccaccactg agggttgtct tgttgcctca accatgcgag
     2641 gttgcaaggc catcaacgcc ggtggcggtg ttaccactgt gcttactcag gacggtatga
     2701 cacgaggtcc ttgtgtttcc ttcccctctc tcaagcgggc tggagccgct aagatctggc
     2761 ttgattccga ggagggtctc aagtccatgc gaaaggcctt caactccacc tctcgatttg
     2821 ctcgtctcca gtctcttcac tctacccttg ctggtaacct gctgtttatt cgattccgaa
     2881 ccaccactgg tgatgccatg ggcatgaaca tgatctccaa gggcgtcgaa cactctctgg
     2941 ccgtcatggt caaggagtac ggcttccctg atatggacat tgtgtctgtc tcgggtaact
     3001 actgcactga caagaagccc gcagcgatca actggatcga aggccgaggc aagagtgttg
     3061 ttgccgaagc caccatccct gctcacattg tcaagtctgt tctcaaaagt gaggttgacg
     3121 ctcttgttga gctcaacatc agcaagaatc tgatcggtag tgccatggct ggctctgtgg
     3181 gaggtttcaa tgcacacgcc gcaaacctgg tgaccgccat ctaccttgcc actggccagg
     3241 atcctgctca gaatgtcgag tcttccaact gcatcacgct gatgagcaac gtcgacggta
     3301 acctgctcat ctccgtttcc atgccttcta tcgaggtcgg taccattggt ggaggtacta
     3361 ttttggagcc ccagggggct atgctggaga tgcttggcgt gcgaggtcct cacatcgaga
     3421 cccccggtgc caacgcccaa cagcttgctc gcatcattgc ttctggagtt cttgcagcgg
     3481 agctttcgct gtgttctgct cttgctgccg gccatcttgt gcaaagtcat atgacccaca
     3541 accggtccca ggctcctact ccggccaagc agtctcaggc cgatctgcag cgtctacaaa
     3601 acggttcgaa tatttgcata cggtcataga tcgcgtgggg gatcctctag agtcgacctg
     3661 caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3721 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3781 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3841 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3901 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3961 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4021 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4081 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4141 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4201 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4261 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4321 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4381 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4441 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4501 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4561 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4621 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4681 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4741 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4801 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4861 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4921 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4981 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5041 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5101 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5161 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5221 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5281 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5341 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5401 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5461 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5521 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5581 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5641 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5701 aatactcat
//
