LOCUS       sequence-205318-        6619 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_4, USER site for 
            cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6619)
  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 6619)
  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: pCfB4787
FEATURES             Location/Qualifiers
     source          1..6619
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     71..88
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     186..203
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     187..203
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    673..706
                     /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)"
     protein_bind    2582..2615
                     /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(3733..3751)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    3950..3983
                     /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(4548..4581)
                     /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(4788..4807)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4813..4832)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4874..4965
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4966..5034,5035..5826)
                     /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(5184..5203)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5948..6536
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6437..6456
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
       61 gaccgagcgc agcgagtcag tgagcgagga agcggaaggc gagagtaggg aactgccagg
      121 catcaaacta agcagaaggc ccctgacgga tggccttttt gcgtttctac aaactctttc
      181 tgtgttgtaa aacgacggcc agtcttaagc tcgggccccc tgggcggttc tgataacgag
      241 taatcgttaa tccgcaaata acgtaaaaac ccgcttcggc gggttttttt atggggggag
      301 tttagggaaa gagcatttgt cagaatattt aagggcgcct gtcactttgc ttgatatatg
      361 agaattattt aaccttataa atgagaaaaa agcaacgcac tttaaataag atacgttgct
      421 ttttcgattg atgaacacct ataattaaac tattcatcta ttatttatga ttttttgtat
      481 atacaatatt tctagtttgt taaagagaat taagaaaata aatctcgaaa ataataaagg
      541 gaaaatcagt ttttgatatc aaaattatac atgtcaacga taatacaaaa tataatacaa
      601 actataagat gttatcagta tttattatgc atttagaata aattttgtgt cgcccttatt
      661 cgactcacta tagaagttcc tattctctag aaagtatagg aacttcactt cattttattt
      721 aaatttgcgg ccgcgaatgc gtgcgatgcg gaggagcaat agacatacga tttgacattt
      781 tgacatttca cagtatcggc agccttgcta tagcaagtgt gtatttgtag atagagacag
      841 gcaattagat ttggagggga ccgcaagaaa cagatcatca ttctgtcggc ccctgttgtc
      901 cctgtggctc tgttagtccg ctggtccgtg gccggagctt gtcatgtttc tggaataccc
      961 tctggccaca ttaactccgc taaagcctca aggccgtaaa ggcgccagtg gcatgttgca
     1021 agcaaagccg cacgcataac acgccacatc acgtgagcgg tcataggccg gtcgccagac
     1081 ggctacaacc ctattacgcg ggtgctctgg ccaaacagac cgtggaggaa ctcttcgaca
     1141 cttgatatta aactctatgc cacttatcgg gcctactgat cagactcaag gtctattgtt
     1201 ctgagagagg aagtttgggg agaaagaacg ggcacatgtg ttgatctcca tcaccgtggg
     1261 gaggggaacg tgcaacgctt acgcaactaa catgaatgaa tacgatatac atcaaagact
     1321 atgatacgca gtattgcaca ctgtacgagt aagggcacta gccactgcac tcaagtgaaa
     1381 ccgttgcccg ggtacgagta tgagtatgta cagtatgttt agtattgtac ttggacaatg
     1441 cttgtatcgt acattctcaa gtgtcaaaca taaatatccg ttgctatatc ctcgcaccac
     1501 cacgtagctc gctatatccc tgtgttgaat ccatccatct tggattgcca attgtgcaca
     1561 cagaaccggg cactcacttc cccatccaca cttcaacgga atgcgtgcga tcgcgtgcat
     1621 tccttctgtt cggaatcaac ctcaaggtta acggccacga tcccctcgtt gttactcttg
     1681 gtcagcccat tgtcggtaac gctggctttg ctaactgggt cgataaactc ttctttggcc
     1741 aggagaaccc cgatgtctcc aaggtgtcca aagaccgaaa gctctaccga atcacccacc
     1801 gaggagatat cgtccctcaa gtgcccttct gggacggtta ccagcactgc tctggtgagg
     1861 tctttattga ctggcccctg atccaccctc ctctctccaa cgttgtcatg tgccagggcc
     1921 agagcaataa acagtgctct gccggtaaca ctctgctcca gcaggtcaat gtgattggaa
     1981 accatctgca gtacttcgtc accgagggtg tctgtggtat ctaagctatt tatcactctt
     2041 tacaacttct acctcaacta tctactttaa taaatgaata tcgtttattc tctatgatta
     2101 ctgtatatgc gttcctctaa gacaaatcga aaccagcatg cgatcgaatg gcatacaaaa
     2161 gtttcttccg aagttgatca atgtcctgat agtcaggcag cttgagaaga ttgacacagg
     2221 tggaggccgt agggaaccga tcaacctgtc taccagcgtt acgaatggca aatgacgggt
     2281 tcaaagcctt gaatccttgc aatggtgcct tggatactga tgtcacaaac ttaagaagca
     2341 gccgcttgtc ctcttcctcg aaactctcaa acacagtcca gaagtccttt atagtttgat
     2401 ctgtatccag atagcctccg taattggtgt gtgtcttcaa atcccagacg tccacattgg
     2461 catgtcctcc actgataagc atttgaagtt catctgcgtt gaacattgag acccacgaag
     2521 ggtcaatgag ctggtataga ccgcccaaga atgcatctga gaacagctga agcttcgtac
     2581 gataacttcg tataatgtat gctatacgaa gttatagaga ccgggttggc ggcgcatttg
     2641 tgtcccaaaa aacagcccca attgccccaa ttgaccccaa attgacccag tagcgggccc
     2701 aaccccggcg agagccccct tctccccaca tatcaaacct cccccggttc ccacacttgc
     2761 cgttaagggc gtagggtact gcagtctgga atctacgctt gttcagactt tgtactagtt
     2821 tctttgtctg gccatccggg taacccatgc cggacgcaaa atagactact gaaaattttt
     2881 ttgctttgtg gttgggactt tagccaaggg tataaaagac caccgtcccc gaattacctt
     2941 tcctcttctt ttctctctct ccttgtcaac tcacacccga aatcgttaag catttccttc
     3001 tgagtataag aatcattcaa aatggtgagt ttcagaggca gcagcaattg ccacgggctt
     3061 tgagcacacg gccgggtgtg gtcccattcc catcgacaca agacgccacg tcatccgacc
     3121 agcacttttt gcagtactaa ccgcagggta ctactttgga tgatactgct tacagataca
     3181 gaacttctgt tccaggtgat gctgaagcta ttgaagcttt ggatggttct ttcactactg
     3241 acactgtttt cagagttact gctactggtg atggtttcac tttgagagaa gttccagttg
     3301 atccaccatt gactaaggtt tttccagatg atgaatccga tgacgaatct gatgatggtg
     3361 aagatggtga tccagattct agaacttttg ttgcttatgg tgatgacggt gacttggctg
     3421 gttttgttgt tgtttcttat tctggttgga atagaagatt gaccgtcgaa gatattgaag
     3481 ttgctccaga acatagaggt catggtgttg gtagagcttt gatgggtttg gctactgaat
     3541 ttgctagaga aagaggtgct ggtcatttgt ggttggaagt tactaatgtt aacgctccag
     3601 ctattcatgc ctatagaaga atgggtttta ccttgtgtgg tttggatact gcattatacg
     3661 atggtactgc atcagatggt gaacaagcct tgtatatgtc tatgccatgt ccatgatcat
     3721 gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa
     3781 ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt
     3841 attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc
     3901 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga taacttcgta
     3961 taatgtatgc tatacgaagt tatgtgatat cagatccact agtggcctca ccgagggata
     4021 gggaacaccg cggaacaggg aacatcgact ctgagacaga ggtgacctga cgagcaacgt
     4081 atgcgagcaa ctcgtatctg cagttggttg gcctggtcac tcgattaatt ggccatccga
     4141 aagcaccatt agcctatcta acgcactaaa atggtgggga agtcaaagga ccatggttcg
     4201 ttcattgttg gctaacggcc cgaaatccgc caaagtgtgg gaaaaaaaca taaaaaatat
     4261 ataaaaaaaa gttgctttgg attttgggat gcagcaacca aaaactcgcg caaaagcccg
     4321 ctcgagtgtc tctgaacgca aatacgaccg acgcggtctc caacatctcg tcgcggtccc
     4381 ctcccccctt cagccagcgt ccctccagac ccaggccaca gcataatatt tcagaccgtt
     4441 gacacccaga gcgggggagc tcgtaataag gtacgcctgt gcagctgggc agtacggtcc
     4501 agtgttaaat cgcgtgcatt cgcggccgca tttaaatccc cacttcagaa gttcctatac
     4561 tttctagaga ataggaactt cactatagag tcgaataagg gcgacacccc ataattagcc
     4621 cgggcgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tggcagttcc
     4681 ctactctcgc atggggagtc cccacactac catcggcgct acggcgtttc acttctgagt
     4741 tcggcatggg gtcaggtggg accaccgcgc tactgccgcc aggcaaacaa ggggtgttat
     4801 gagccatatt caggtataaa tgggctcgcg ataatgttca gaattggtta attggttgta
     4861 acactgaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4921 caataaccct gataaatgct tcaataatat tgaaaaagga agaatatgag tattcaacat
     4981 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5041 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5101 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5161 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5221 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5281 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5341 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5401 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5461 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcgatggca
     5521 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5581 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5641 ggctggttta ttgctgataa atccggagcc ggtgagcgtg gttctcgcgg tatcatcgca
     5701 gcgctggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5761 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5821 tggtaagcag agcattacgc tgacttgacg ggacggcgca agctcatgac caaaatccct
     5881 taacgtgagt tacgcgcgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5941 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6001 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6061 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttagccc
     6121 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6181 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6241 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6301 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6361 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6421 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6481 tctgacttga gcgtcaattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6541 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6601 ttcctgcgtt atcccctga
//
