LOCUS       sequence-248370-        7114 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing integration cassette at XI-3 integration site in 
            Saccharomyces cerevisiae for overexpression of tHMG1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7114)
  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 7114)
  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: pCfB8383
FEATURES             Location/Qualifiers
     source          1..7114
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1408..1419
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     terminator      2017..2206
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2222..2241)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2223..2241)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2251..2267)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2260..2282)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2906..2939)
                     /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(3409..3425)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3524..3541)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3695..4283)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3775..3794)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4405..5196,5197..5265))
                     /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     5028..5047
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5266..5357)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5399..5418
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     protein_bind    5650..5683
                     /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)"
     terminator      complement(6335..6500)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6421..6445
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
ORIGIN
        1 gtcctagcga cggctcacag gttttgtaac aagcaatcga aggttctgga atggcgggaa
       61 agggtttagt accacatgct atgatgccca ctgtgatctc cagagcaaag ttcgttcgat
      121 cgtactgtta ctctctctct ttcaaacaga attgtccgaa tcgtgtgaca acaacagcct
      181 gttctcacac actcttttct tctaaccaag ggggtggttt agtttagtag aacctcgtga
      241 aacttacatt tacatatata taaacttgca taaattggtc aatgcaagaa atacatattt
      301 ggtcttttct aattcgtagt ttttcaagtt cttagatgct ttctttttct cttttttaca
      361 gatcatcaag gaagtaatta tctacttttt acaacaaata taaaacaaat ctgtcataaa
      421 acaatgactg cagaccaatt ggtgaagact gaagtcacca agaagtcttt tactgctcct
      481 gtacaaaagg cttctacacc agttttaacc aataaaacag tcatttctgg atcgaaagtc
      541 aaaagtttat catctgcgca atcgagctca tcaggacctt catcatctag tgaggaagat
      601 gattcccgcg atattgaaag cttggataag aaaatacgtc ctttagaaga attagaagca
      661 ttattaagta gtggaaatac aaaacaattg aagaacaaag aggtcgctgc cttggttatt
      721 cacggtaagt tacctttgta cgctttggag aaaaaattag gtgatactac gagagcggtt
      781 gcggtacgta ggaaggctct ttcaattttg gcagaagctc ctgtattagc atctgatcgt
      841 ttaccatata aaaattatga ctacgaccgc gtatttggcg cttgttgtga aaatgttata
      901 ggttacatgc ctttgcccgt tggtgttata ggccccttgg ttatcgatgg tacatcttat
      961 catataccaa tggcaactac agagggttgt ttggtagctt ctgccatgcg tggctgtaag
     1021 gcaatcaatg ctggcggtgg tgcaacaact gttttaacta aggatggtat gacaagaggc
     1081 ccagtagtcc gtttcccaac tttgaaaaga tctggtgcct gtaagatatg gttagactca
     1141 gaagagggac aaaacgcaat taaaaaagct tttaactcta catcaagatt tgcacgtctg
     1201 caacatattc aaacttgtct agcaggagat ttactcttca tgagatttag aacaactact
     1261 ggtgacgcaa tgggtatgaa tatgatttct aagggtgtcg aatactcatt aaagcaaatg
     1321 gtagaagagt atggctggga agatatggag gttgtctccg tttctggtaa ctactgtacc
     1381 gacaaaaaac cagctgccat caactggatc gaaggtcgtg gtaagagtgt cgtcgcagaa
     1441 gctactattc ctggtgatgt tgtcagaaaa gtgttaaaaa gtgatgtttc cgcattggtt
     1501 gagttgaaca ttgctaagaa tttggttgga tctgcaatgg ctgggtctgt tggtggattt
     1561 aacgcacatg cagctaattt agtgacagct gttttcttgg cattaggaca agatcctgca
     1621 caaaatgtcg aaagttccaa ctgtataaca ttgatgaaag aagtggacgg tgatttgaga
     1681 atttccgtat ccatgccatc catcgaagta ggtaccatcg gtggtggtac tgttctagaa
     1741 ccacaaggtg ccatgttgga cttattaggt gtaagaggcc cacatgctac cgctcctggt
     1801 accaacgcac gtcaattagc aagaatagtt gcctgtgccg tcttggcagg tgaattatcc
     1861 ttatgtgctg ccctagcagc cggccatttg gttcaaagtc atatgaccca caacaggaaa
     1921 cctgctgaac caacaaaacc taacaatttg gacgccactg atataaatcg tttgaaagat
     1981 gggtccgtca cctgcattaa atcctgaatc gcgtgtatcc gctctaaccg aaaaggaagg
     2041 agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag
     2101 aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa
     2161 cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaagatcg cgtccaattc
     2221 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg
     2281 ggaaaaccct ggcgttaccc ctgcaggact agtgctgagg cattaattta cgtggattga
     2341 gccagcaata cagatcatta ttaaattgtt ttgtacatga tgttagtata taatcgtaaa
     2401 gcttttctaa tatgtatacc ttatacatgg aactccacag aacttgcaaa cataccaaaa
     2461 atcccttatt cttgttcact cattttacat caaaaaataa tatttcagtt attaaggaaa
     2521 ataaaaaaat agattagaga agcattttga agaaatagta tattctttta ttgaacctaa
     2581 gagcgtgata tttttactcg aaataaaata cgaaaaatct atgcactcat ctttccgact
     2641 actattggct cctgctcaaa aaaagaggga aaaaaagctc caaaattcta tcttttccta
     2701 tcgctcctgt cctatcctta ttacgttcat tactatttta atactatcca ttcttttatt
     2761 ttcagtctaa aaaaaacatt tctcataacg ggaaaagcaa aaaaatgtca agcttataca
     2821 tcaaaacacc actgcatgca ttatctgctg gtccggattc tcaattaaga cctcagcgcg
     2881 gccgcaaatt taaataaaat gaagtgaagt tcctatactt tctagagaat aggaacttct
     2941 atagtgagtc gaataagggc gacacaaaat ttattctaaa tgcataataa atactgataa
     3001 catcttatag tttgtattat attttgtatt atcgttgaca tgtataattt tgatatcaaa
     3061 aactgatttt ccctttatta ttttcgagat ttattttctt aattctcttt aacaaactag
     3121 aaatattgta tatacaaaaa atcataaata atagatgaat agtttaatta taggtgttca
     3181 tcaatcgaaa aagcaacgta tcttatttaa agtgcgttgc ttttttctca tttataaggt
     3241 taaataattc tcatatatca agcaaagtga caggcgccct taaatattct gacaaatgct
     3301 ctttccctaa actcccccca taaaaaaacc cgccgaagcg ggtttttacg ttatttgcgg
     3361 attaacgatt actcgttatc agaaccgccc agggggcccg agcttaagac tggccgtcgt
     3421 tttacaacac agaaagagtt tgtagaaacg caaaaaggcc atccgtcagg ggccttctgc
     3481 ttagtttgat gcctggcagt tccctactct cgccttccgc ttcctcgctc actgactcgc
     3541 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3601 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3661 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3721 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3781 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3841 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3901 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3961 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4021 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4081 taggcggtgc tacagagttc ttgaagtggt gggctaacta cggctacact agaagaacag
     4141 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4201 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4261 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4321 agtggaacga cgcgcgcgta actcacgtta agggattttg gtcatgagct tgcgccgtcc
     4381 cgtcaagtca gcgtaatgct ctgcttacca atgcttaatc agtgaggcac ctatctcagc
     4441 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4501 acgggagggc ttaccatctg gccccagcgc tgcgatgata ccgcgagaac cacgctcacc
     4561 ggctccggat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4621 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4681 ttcgccagtt aatagtttgc gcaacgttgt tgccatcgct acaggcatcg tggtgtcacg
     4741 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4801 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4861 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4921 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4981 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5041 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5101 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5161 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5221 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatattct tcctttttca
     5281 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5341 ttagaaaaat aaacaaatag gggtcagtgt tacaaccaat taaccaattc tgaacattat
     5401 cgcgagccca tttatacctg aatatggctc ataacgcccc ttgtttgcct ggcggcagta
     5461 gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg
     5521 gtagtgtggg gactccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     5581 gctcagtcga aagactgggc ctttcgcccg ggctaattat ggggtgtcgc ccttattcga
     5641 ctctatagtg aagttcctat tctctagaaa gtataggaac ttctgaagtg gggatttaaa
     5701 tgcggccgcg ctgagggttt aatagttact tgctctatgc gtttgcgcat cctcttttta
     5761 cttttttttt cagtaaagcc taagcataaa tcgttttata cgtacgacac gttcaacttt
     5821 tcttggttag tagtggcaat ctctgcaata catacaggga gtcatggtct atcatcttgt
     5881 ccaatcaaag aagcatcggt tcagatcgag caaactgtag ggagaaagga aagtagaaat
     5941 gcagagtgtg ctatatgtcc aatctcggtt ttgtagtttg gatgtcatta gagatctacc
     6001 acccaaccgg ctgctttcat gtggaacaga aaagaaatcg gggcgcttcc tcttctgtat
     6061 tcctttagtt aacgttttta ttcagccatc taaccatcat acccccatac ggtaacaaaa
     6121 cctcttctaa gaaaagaagt ctctgctcct ccgccatctt atttttattc gctgcgcgcg
     6181 tttattgtcg catcgctggc cagcaaaaaa ttggttgcct ttttttacct aaaaaagaca
     6241 catctaactg attagttttc cgttttagga tattgacgcc aagcgtgcgt ctgattatta
     6301 agtcctcagc gagctcgcat ggaatgcgtg cgatgagcga cctcatgcta tacctgagaa
     6361 agcaacctga cctacaggaa agagttactc aagaataaga attttcgttt taaaacctaa
     6421 gagtcacttt aaaatttgta tacacttatt ttttttataa cttatttaat aataaaaatc
     6481 ataaatcata agaaattcgc ttatttagaa gtgtcaacaa cgtatctaca cgtgcgatgg
     6541 aagtaccttc aaagaatggg gtcttatctt gttttgcaag taccactgag caggataata
     6601 atagaaatga taatatacta tagtagagat aacgtcgatg acttcccata ctgtaattgc
     6661 ttttagttgt gtatttttag tgtgcaagtt tctgtaaatc gattaatttt tttttctttc
     6721 ctctttttat taaccttaat ttttatttta gattcctgac ttcaactcaa gacgcacaga
     6781 tattataaca tctgcataat aggcatttgc aagaattact cgtgagtaag gaaagagtga
     6841 ggaactatcg catacctgca tttaaagatg ccgatttggg cgcgaatcct ttattttggc
     6901 ttcaccctca tactattatc agggccagaa aaaggaagtg tttccctcct tcttgaattg
     6961 atgttaccct cataaagcac gtggcctctt atcgagaaag aaattaccgt cgctcgtgat
     7021 ttgtttgcaa aaagaacaaa actgaaaaaa cccagacacg ctcgacttcc tgtcttccta
     7081 ttgattgcag cttccaattt cgtcacacaa caag
//
