LOCUS       sequence-205316-        6562 bp    DNA     circular SYN 14-APR-2025
DEFINITION  EasyCloneYALI system-based yeast integrative vector carrying 
            loxP-flanked nourseothricin-resistance marker, integration into 
            Yarrowia lipolytica chromosomal location IntC_3, USER site for 
            cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6562)
  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 6562)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pCfB4786
FEATURES             Location/Qualifiers
     source          1..6562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    21..54
                     /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    1913..1946
                     /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(3064..3082)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    3281..3314
                     /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(3839..3872)
                     /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(4079..4098)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4104..4123)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4165..4256
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4257..4325,4326..5117)
                     /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(4475..4494)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5239..5827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5728..5747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5981..5998
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6096..6113
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6097..6113
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cccttattcg actcactata gaagttccta ttctctagaa agtataggaa cttcacttca
       61 ttttatttaa atttgcggcc gcgaatgcgt gcgataaaaa actgtagtag tgtggtgatg
      121 gagtcataac ccgctccggc tatgtgcgga tacagtattg tctcagacag tggtcgcgag
      181 caatgtcttg tgcatcttca tacttttttt tttttttatt ttgagtgggg aggttgaggt
      241 tgggtcagag gttgggtcat ccatgaagag gttaacagaa cgagccacga tagggggaag
      301 gatcttcaac tcgatccgct attgttctca gtgatggcaa atagattcat ctaagaccta
      361 ccggtacaag taaagtacag cacacgtaat ttttcttgcg gctagtctac tctcagtgca
      421 tgtaacgtgc ggatctagcc catgaacagg tggtttttac cctcctgctc tccacatttc
      481 cagatagatg caggcttcta tggttgttcc acactcttgt cagaggagat gcccgtatac
      541 gaccaccata agcgacccta ttacggggcc cgaacaagcc ccgttgatag acactgtaac
      601 gtgcaacgct tacgcaacta acatgaatga atacgatata catcaaagac tatgatacgc
      661 agtattgcac actgtacgag taagggcact agccactgca ctcaagtgaa accgttgccc
      721 gggtacgagt atgagtatgt acagtatgtt tagtattgta cttggacaat gcttgtatcg
      781 tacattctca agtgtcaaac ataaatatcc gttgctatat cctcgcacca ccacgtagct
      841 cgctatatcc ctgtgttgaa tccatccatc ttggattgcc aattgtgcac acagaaccgg
      901 gcactcactt ccccatccac acttcaacgg aatgcgtgcg atcgcgtgca ttccttctgt
      961 tcggaatcaa cctcaaggtt aacggccacg atcccctcgt tgttactctt ggtcagccca
     1021 ttgtcggtaa cgctggcttt gctaactggg tcgataaact cttctttggc caggagaacc
     1081 ccgatgtctc caaggtgtcc aaagaccgaa agctctaccg aatcacccac cgaggagata
     1141 tcgtccctca agtgcccttc tgggacggtt accagcactg ctctggtgag gtctttattg
     1201 actggcccct gatccaccct cctctctcca acgttgtcat gtgccagggc cagagcaata
     1261 aacagtgctc tgccggtaac actctgctcc agcaggtcaa tgtgattgga aaccatctgc
     1321 agtacttcgt caccgagggt gtctgtggta tctaagctat ttatcactct ttacaacttc
     1381 tacctcaact atctacttta ataaatgaat atcgtttatt ctctatgatt actgtatatg
     1441 cgttcctcta agacaaatcg aaaccagcat gcgatcgaat ggcatacaaa agtttcttcc
     1501 gaagttgatc aatgtcctga tagtcaggca gcttgagaag attgacacag gtggaggccg
     1561 tagggaaccg atcaacctgt ctaccagcgt tacgaatggc aaatgacggg ttcaaagcct
     1621 tgaatccttg caatggtgcc ttggatactg atgtcacaaa cttaagaagc agccgcttgt
     1681 cctcttcctc gaaactctca aacacagtcc agaagtcctt tatagtttga tctgtatcca
     1741 gatagcctcc gtaattggtg tgtgtcttca aatcccagac gtccacattg gcatgtcctc
     1801 cactgataag catttgaagt tcatctgcgt tgaacattga gacccacgaa gggtcaatga
     1861 gctggtatag accgcccaag aatgcatctg agaacagctg aagcttcgta cgataacttc
     1921 gtataatgta tgctatacga agttatagag accgggttgg cggcgcattt gtgtcccaaa
     1981 aaacagcccc aattgcccca attgacccca aattgaccca gtagcgggcc caaccccggc
     2041 gagagccccc ttctccccac atatcaaacc tcccccggtt cccacacttg ccgttaaggg
     2101 cgtagggtac tgcagtctgg aatctacgct tgttcagact ttgtactagt ttctttgtct
     2161 ggccatccgg gtaacccatg ccggacgcaa aatagactac tgaaaatttt tttgctttgt
     2221 ggttgggact ttagccaagg gtataaaaga ccaccgtccc cgaattacct ttcctcttct
     2281 tttctctctc tccttgtcaa ctcacacccg aaatcgttaa gcatttcctt ctgagtataa
     2341 gaatcattca aaatggtgag tttcagaggc agcagcaatt gccacgggct ttgagcacac
     2401 ggccgggtgt ggtcccattc ccatcgacac aagacgccac gtcatccgac cagcactttt
     2461 tgcagtacta accgcagggt actactttgg atgatactgc ttacagatac agaacttctg
     2521 ttccaggtga tgctgaagct attgaagctt tggatggttc tttcactact gacactgttt
     2581 tcagagttac tgctactggt gatggtttca ctttgagaga agttccagtt gatccaccat
     2641 tgactaaggt ttttccagat gatgaatccg atgacgaatc tgatgatggt gaagatggtg
     2701 atccagattc tagaactttt gttgcttatg gtgatgacgg tgacttggct ggttttgttg
     2761 ttgtttctta ttctggttgg aatagaagat tgaccgtcga agatattgaa gttgctccag
     2821 aacatagagg tcatggtgtt ggtagagctt tgatgggttt ggctactgaa tttgctagag
     2881 aaagaggtgc tggtcatttg tggttggaag ttactaatgt taacgctcca gctattcatg
     2941 cctatagaag aatgggtttt accttgtgtg gtttggatac tgcattatac gatggtactg
     3001 catcagatgg tgaacaagcc ttgtatatgt ctatgccatg tccatgatca tgtaattagt
     3061 tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt
     3121 tagacaacct gaagtctagg tccctattta tttttttata gttatgttag tattaagaac
     3181 gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat
     3241 tatactgaaa accttgcttg agaaggtttt gggacgctcg ataacttcgt ataatgtatg
     3301 ctatacgaag ttatgtgata tcagatccac tagtggcctg ccatagcact attgtagagt
     3361 ggccattgtt cgtgtcgatc cgtacgacga tcatggagcg agcggccacg aaattagata
     3421 gccactaaaa ctagtcctgt ccacggaagg ccgtttgtta gccgcttata tcgttggaac
     3481 aatacttttg ttgccggcat ctaatttcat aatttcgtga ttaatttttc ccgccaaaaa
     3541 ataaaaaatc aaaaacaaaa aataaaaagg aacttcggta agagcgctcc cgaaaccgga
     3601 aatgcatggt ttctctcatg tgtgcctctt gcttgtctca ggccggtcca gaaacaaacc
     3661 tcgagcttcc gcctccaaga ccctgctttc ggagaagcag ccaatggctg ttgtttgtca
     3721 ctgttccctg tcttggtttt tttggccctt cttttcttct tccccccatg aaaccttgtg
     3781 tgtgtcctgt gcaataatta tcgcgtgcat tcgcggccgc atttaaatcc ccacttcaga
     3841 agttcctata ctttctagag aataggaact tcactataga gtcgaataag ggcgacaccc
     3901 cataattagc ccgggcgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc
     3961 ctggcagttc cctactctcg catggggagt ccccacacta ccatcggcgc tacggcgttt
     4021 cacttctgag ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaaca
     4081 aggggtgtta tgagccatat tcaggtataa atgggctcgc gataatgttc agaattggtt
     4141 aattggttgt aacactgacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4201 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagaatatga
     4261 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4321 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     4381 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4441 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4501 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     4561 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4621 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     4681 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4741 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4801 tagcgatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4861 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4921 cccttccggc tggctggttt attgctgata aatccggagc cggtgagcgt ggttctcgcg
     4981 gtatcatcgc agcgctgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5041 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5101 tgattaagca ttggtaagca gagcattacg ctgacttgac gggacggcgc aagctcatga
     5161 ccaaaatccc ttaacgtgag ttacgcgcgc gtcgttccac tgagcgtcag accccgtaga
     5221 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5281 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5341 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5401 gtagttagcc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5461 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5521 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5581 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5641 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5701 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5761 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5821 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5881 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5941 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6001 agcggaaggc gagagtaggg aactgccagg catcaaacta agcagaaggc ccctgacgga
     6061 tggccttttt gcgtttctac aaactctttc tgtgttgtaa aacgacggcc agtcttaagc
     6121 tcgggccccc tgggcggttc tgataacgag taatcgttaa tccgcaaata acgtaaaaac
     6181 ccgcttcggc gggttttttt atggggggag tttagggaaa gagcatttgt cagaatattt
     6241 aagggcgcct gtcactttgc ttgatatatg agaattattt aaccttataa atgagaaaaa
     6301 agcaacgcac tttaaataag atacgttgct ttttcgattg atgaacacct ataattaaac
     6361 tattcatcta ttatttatga ttttttgtat atacaatatt tctagtttgt taaagagaat
     6421 taagaaaata aatctcgaaa ataataaagg gaaaatcagt ttttgatatc aaaattatac
     6481 atgtcaacga taatacaaaa tataatacaa actataagat gttatcagta tttattatgc
     6541 atttagaata aattttgtgt cg
//
