LOCUS       sequence-205312-        6603 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast integrative vector carrying 
            loxP-flanked nourseothricin-resistance marker, integration into 
            Yarrowia lipolytica chromosomal location IntE_2, USER site for 
            cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6603)
  AUTHORS   Holkenbrink C, Dam MI, Kildegaard KR, Beder J, Dahlin J, Domenech 
            Belda D, Borodina I
  TITLE     EasyCloneYALI: CRISPR/Cas9-Based Synthetic Toolbox for Engineering 
            of the Yeast Yarrowia lipolytica.
  JOURNAL   Biotechnol J. 2018 Jan 29. doi: 10.1002/biot.201700543.
  PUBMED    29377615
REFERENCE   2  (bases 1 to 6603)
  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: "Biotechnol 
            J. 2018 Jan 29. doi: 10.1002/biot.201700543."
COMMENT     Sequence Label: pCfB4779
FEATURES             Location/Qualifiers
     source          1..6603
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1815..1848
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2966..2984)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    3183..3216
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(3785..3818)
                     /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(4025..4044)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4050..4069)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4111..4202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4203..4271,4272..5063)
                     /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(4421..4440)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5185..5773
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5674..5693
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5927..5944
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6042..6059
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6043..6059
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6529..6562
                     /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)"
ORIGIN
        1 atggtgccaa atcgcttgtc actttgacac aatgaagata ttcgattcag tcgataccat
       61 ggccgtcaac tttcttgtcc gacctctcaa ctttgtatct atgtctcagg acggagtgtt
      121 ggtccatcca gcccctacca gttccgtggt gatgttgaaa gggctatttt tatccagatc
      181 agagggtcac tggagcttga ataggatcca caagaagttc aaaggacttt actggtgttt
      241 cttttgtccg agaacttcaa caactccgtc tggtgtctcc aagatagatt ctggtgctct
      301 ccacgcctat acatctccaa gtactgtaca ccacaaacac cttccgggtg gttgcttaca
      361 gtttcaatag gcgaccgctt gatttcggca aatctacacc tgccagctac gcatattgca
      421 ggaagcagca tcatggaatg gactgtatag ccataagaca agaatgctct ggagaaatcg
      481 tccgattttg ctgcactaag acgtgcaacg cttacgcaac taacatgaat gaatacgata
      541 tacatcaaag actatgatac gcagtattgc acactgtacg agtaagggca ctagccactg
      601 cactcaagtg aaaccgttgc ccgggtacga gtatgagtat gtacagtatg tttagtattg
      661 tacttggaca atgcttgtat cgtacattct caagtgtcaa acataaatat ccgttgctat
      721 atcctcgcac caccacgtag ctcgctatat ccctgtgttg aatccatcca tcttggattg
      781 ccaattgtgc acacagaacc gggcactcac ttccccatcc acacttcaac ggaatgcgtg
      841 cgatcgcgtg cattccttct gttcggaatc aacctcaagg ttaacggcca cgatcccctc
      901 gttgttactc ttggtcagcc cattgtcggt aacgctggct ttgctaactg ggtcgataaa
      961 ctcttctttg gccaggagaa ccccgatgtc tccaaggtgt ccaaagaccg aaagctctac
     1021 cgaatcaccc accgaggaga tatcgtccct caagtgccct tctgggacgg ttaccagcac
     1081 tgctctggtg aggtctttat tgactggccc ctgatccacc ctcctctctc caacgttgtc
     1141 atgtgccagg gccagagcaa taaacagtgc tctgccggta acactctgct ccagcaggtc
     1201 aatgtgattg gaaaccatct gcagtacttc gtcaccgagg gtgtctgtgg tatctaagct
     1261 atttatcact ctttacaact tctacctcaa ctatctactt taataaatga atatcgttta
     1321 ttctctatga ttactgtata tgcgttcctc taagacaaat cgaaaccagc atgcgatcga
     1381 atggcataca aaagtttctt ccgaagttga tcaatgtcct gatagtcagg cagcttgaga
     1441 agattgacac aggtggaggc cgtagggaac cgatcaacct gtctaccagc gttacgaatg
     1501 gcaaatgacg ggttcaaagc cttgaatcct tgcaatggtg ccttggatac tgatgtcaca
     1561 aacttaagaa gcagccgctt gtcctcttcc tcgaaactct caaacacagt ccagaagtcc
     1621 tttatagttt gatctgtatc cagatagcct ccgtaattgg tgtgtgtctt caaatcccag
     1681 acgtccacat tggcatgtcc tccactgata agcatttgaa gttcatctgc gttgaacatt
     1741 gagacccacg aagggtcaat gagctggtat agaccgccca agaatgcatc tgagaacagc
     1801 tgaagcttcg tacgataact tcgtataatg tatgctatac gaagttatag agaccgggtt
     1861 ggcggcgcat ttgtgtccca aaaaacagcc ccaattgccc caattgaccc caaattgacc
     1921 cagtagcggg cccaaccccg gcgagagccc ccttctcccc acatatcaaa cctcccccgg
     1981 ttcccacact tgccgttaag ggcgtagggt actgcagtct ggaatctacg cttgttcaga
     2041 ctttgtacta gtttctttgt ctggccatcc gggtaaccca tgccggacgc aaaatagact
     2101 actgaaaatt tttttgcttt gtggttggga ctttagccaa gggtataaaa gaccaccgtc
     2161 cccgaattac ctttcctctt cttttctctc tctccttgtc aactcacacc cgaaatcgtt
     2221 aagcatttcc ttctgagtat aagaatcatt caaaatggtg agtttcagag gcagcagcaa
     2281 ttgccacggg ctttgagcac acggccgggt gtggtcccat tcccatcgac acaagacgcc
     2341 acgtcatccg accagcactt tttgcagtac taaccgcagg gtactacttt ggatgatact
     2401 gcttacagat acagaacttc tgttccaggt gatgctgaag ctattgaagc tttggatggt
     2461 tctttcacta ctgacactgt tttcagagtt actgctactg gtgatggttt cactttgaga
     2521 gaagttccag ttgatccacc attgactaag gtttttccag atgatgaatc cgatgacgaa
     2581 tctgatgatg gtgaagatgg tgatccagat tctagaactt ttgttgctta tggtgatgac
     2641 ggtgacttgg ctggttttgt tgttgtttct tattctggtt ggaatagaag attgaccgtc
     2701 gaagatattg aagttgctcc agaacataga ggtcatggtg ttggtagagc tttgatgggt
     2761 ttggctactg aatttgctag agaaagaggt gctggtcatt tgtggttgga agttactaat
     2821 gttaacgctc cagctattca tgcctataga agaatgggtt ttaccttgtg tggtttggat
     2881 actgcattat acgatggtac tgcatcagat ggtgaacaag ccttgtatat gtctatgcca
     2941 tgtccatgat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg
     3001 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta
     3061 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca
     3121 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct
     3181 cgataacttc gtataatgta tgctatacga agttatgtga tatcagatcc actagtggcc
     3241 tgtgtggatt gcgatattga agtgttgact cgaagagaca gctgcctgtc aagttgattg
     3301 ttagctcctt ggcatacaga tgcaagaaat gagagtggcc atacggcggc agttagcata
     3361 agatactact tgtaggagag agcgctcacc accagctaaa tttaggaccc tggctgtcta
     3421 tttacatttt tgcaccctgt tggctgccta gctatgcggc cagtcagccc gctggacatg
     3481 tgtttggcat gtgtttggca tgtgtttggt gcagggagaa ccgcgatggt gaccgtccaa
     3541 gcaattaccc gtttggacca gaagagtaaa gagcgacatt tgcaaaagaa acgcgctgaa
     3601 aaagcaactc cagcgatgag gaactgagag tctgaagtct ctggacgagg agtgggaggg
     3661 gggatgcctt ctgtgggtgc cgtacgtgtc taccgagaag ttaacaaccc tcctgttggt
     3721 atagtctaca aacctggcgt atttcatcgc gtgcattcgc ggccgcattt aaatccccac
     3781 ttcagaagtt cctatacttt ctagagaata ggaacttcac tatagagtcg aataagggcg
     3841 acaccccata attagcccgg gcgaaaggcc cagtctttcg actgagcctt tcgttttatt
     3901 tgatgcctgg cagttcccta ctctcgcatg gggagtcccc acactaccat cggcgctacg
     3961 gcgtttcact tctgagttcg gcatggggtc aggtgggacc accgcgctac tgccgccagg
     4021 caaacaaggg gtgttatgag ccatattcag gtataaatgg gctcgcgata atgttcagaa
     4081 ttggttaatt ggttgtaaca ctgaccccta tttgtttatt tttctaaata cattcaaata
     4141 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4201 atatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4261 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4321 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4381 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4441 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4501 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4561 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4621 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4681 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4741 tgcctgtagc gatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4801 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4861 gctcggccct tccggctggc tggtttattg ctgataaatc cggagccggt gagcgtggtt
     4921 ctcgcggtat catcgcagcg ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4981 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5041 cctcactgat taagcattgg taagcagagc attacgctga cttgacggga cggcgcaagc
     5101 tcatgaccaa aatcccttaa cgtgagttac gcgcgcgtcg ttccactgag cgtcagaccc
     5161 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5221 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5281 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     5341 gtagccgtag ttagcccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5401 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5461 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5521 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5581 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5641 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5701 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5761 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5821 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5881 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5941 cgaggaagcg gaaggcgaga gtagggaact gccaggcatc aaactaagca gaaggcccct
     6001 gacggatggc ctttttgcgt ttctacaaac tctttctgtg ttgtaaaacg acggccagtc
     6061 ttaagctcgg gccccctggg cggttctgat aacgagtaat cgttaatccg caaataacgt
     6121 aaaaacccgc ttcggcgggt ttttttatgg ggggagttta gggaaagagc atttgtcaga
     6181 atatttaagg gcgcctgtca ctttgcttga tatatgagaa ttatttaacc ttataaatga
     6241 gaaaaaagca acgcacttta aataagatac gttgcttttt cgattgatga acacctataa
     6301 ttaaactatt catctattat ttatgatttt ttgtatatac aatatttcta gtttgttaaa
     6361 gagaattaag aaaataaatc tcgaaaataa taaagggaaa atcagttttt gatatcaaaa
     6421 ttatacatgt caacgataat acaaaatata atacaaacta taagatgtta tcagtattta
     6481 ttatgcattt agaataaatt ttgtgtcgcc cttattcgac tcactataga agttcctatt
     6541 ctctagaaag tataggaact tcacttcatt ttatttaaat ttgcggccgc gaatgcgtgc
     6601 gat
//
