LOCUS       sequence-472125-        7115 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The base plasmid of TUNEYALI forTF09.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7115)
  TITLE     Regulating transcription factors in Yarrowia lipolytica
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7115)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCfb13199
FEATURES             Location/Qualifiers
     source          1..7115
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        459..533
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(940..973)
                     /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(1180..1199)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1205..1224)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1266..1357
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1358..1426,1427..2218)
                     /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(1576..1595)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2340..2928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2829..2848
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3082..3099
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3197..3214
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3198..3214
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3684..3717
                     /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)"
     CDS             4859..5887
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4859..5887
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     primer_bind     complement(5904..5922)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 ttcgtagata atggaataca aatggatatc cagagtatac acatggatag tatacactga
       61 cacgacaatt ctgtatctct ttatgttaac tactgtgagg cattaaatag agcttgatat
      121 ataaaatgtt acatttcaca gtctgaactt ttgcagatta cctaatttgg taagatatta
      181 attatgaact gaaagttgat ggcatcccta aatttgatgc aacggaatgc gtgcgatagt
      241 gaatcattgc taacagatca agtaatcgta tcaatcgttc cacgtcttat ccagactcga
      301 tttccgccct aacctaagga aactgctgag taatttcgag gttggttcct aacaaaagcc
      361 aagaaacgcg ccgatggttt agtggtaaaa tccatcgttg ccatcgatgg gcccccggtt
      421 cgattccggg tcggcgcatt gtggacgatg ctataagggt tttagagcta gaaatagcaa
      481 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtggtgcttt
      541 ttttgtgtgg gtacggttga ctgctgccat tctgcttaag cagaacgccg actggggtct
      601 tgagactgcc attggcgcct ccaccgttct tcttggagcc tccattcccc ggatcatcaa
      661 ggccggtatc aagcctgtcc ctgtgggtct cgccgtgctt ggttctctta acttgggatg
      721 actgaagagc tcttcaatgg tgaaaaccat ctacatcacc cgacacgggt tccgagccaa
      781 ctggctgacc gaccagccca ttcccccgtc tcccaccggt atcgagagtg atcctgctct
      841 tgctgagaag ggagttgtcc aggccaagga gctaggtgaa tatctcaagg acatcaagat
      901 cgcgtgcatt cgcggccgca nntttaaatm mmmwswwswg aagttcctat actttctaga
      961 gaataggaac ttcwmtatag agtcgaataa gggcgacacc ccataattag cccgggcgaa
     1021 aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc
     1081 gcatggggag tccccacact accatcggcg ctacggcgtt tcacttctga gttcggcatg
     1141 gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac aaggggtgtt atgagccata
     1201 ttcaggtata aatgggctcg cgataatgtt cagaattggt taattggttg taacactgac
     1261 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1321 ctgataaatg cttcaataat attgaaaaag gaagaatatg agtattcaac atttccgtgt
     1381 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     1441 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     1501 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     1561 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     1621 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     1681 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     1741 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     1801 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     1861 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg caacaacgtt
     1921 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     1981 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     2041 tattgctgat aaatccggag ccggtgagcg tggttctcgc ggtatcatcg cagcgctggg
     2101 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     2161 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaagc
     2221 agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga
     2281 gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2341 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2401 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2461 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc ccaccacttc
     2521 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2581 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2641 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2701 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2761 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2821 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2881 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2941 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3001 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3061 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaagg cgagagtagg
     3121 gaactgccag gcatcaaact aagcagaagg cccctgacgg atggcctttt tgcgtttcta
     3181 caaactcttt ctgtgttgta aaacgacggc cagtcttaag ctcgggcccc ctgggcggtt
     3241 ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa cccgcttcgg cgggtttttt
     3301 tatgggggga gtttagggaa agagcatttg tcagaatatt taagggcgcc tgtcactttg
     3361 cttgatatat gagaattatt taaccttata aatgagaaaa aagcaacgca ctttaaataa
     3421 gatacgttgc tttttcgatt gatgaacacc tataattaaa ctattcatct attatttatg
     3481 attttttgta tatacaatat ttctagtttg ttaaagagaa ttaagaaaat aaatctcgaa
     3541 aataataaag ggaaaatcag tttttgatat caaaattata catgtcaacg ataatacaaa
     3601 atataataca aactataaga tgttatcagt atttattatg catttagaat aaattttgtg
     3661 tcgcccttat tcgactcact atagaagttc ctattctcta gaaagtatag gaacttcact
     3721 tcattttatt taaatttgcg gccgctatgt ctgataaaag gatgtaacat aggcaagctg
     3781 ctcgtgagtg ttgagtacga accttagatc caaatcaccc gcacccacgg atatacttgc
     3841 ttgaatatac agtagtaagg agtttggcgc ccgttttttc gagccccaca cgtttcggtg
     3901 agtatgagcg gcggcagatt cgagcgtttc cggtttccgc ggctggacga gagcccatga
     3961 tgggggctcc caccaccagc aatcagggcc ctgattacac acccacctgt aatgtcatgc
     4021 tgttcatcgt ggttaatgct gctgtgtgct gtgtgtgtgt gttgtttggc gctcattgtt
     4081 gcgttatgca gcgtacacca caatattgga agcttattag cctttctatt ttttcgtttg
     4141 caaggcttaa caacattgct gtggagaggg atggggatat ggaggccgct ggagggagtc
     4201 ggagaggcgt tttggagcgg cttggcctgg cgcccagctc gcgaaacgca cctaggaccc
     4261 tttggcacgc cgaaatgtgc cacttttcag tctagtaacg ccttacctac gtcattccat
     4321 gcatgcatgt ttgcgccttt tttcccttgc ccttgatcgc cacacagtac agtgcactgt
     4381 acagtggagg ttttgggggg gtcttagatg ggagctaaaa gcggcctagc ggtacactag
     4441 tgggattgta tggagtggca tggagcctag gtggagcctg acaggacgca cgaccggcta
     4501 gcccgtgaca gacgatgggt ggctcctgtt gtccaccgcg tacaaatgtt tgggccaaag
     4561 tcttgtcagc cttgcttgcg aacctaattc ccaattttgt cacttcgcac ccccattgat
     4621 cgagccctaa cccctgccca tcaggcaatc caattaagct cgcattgtct gccttgttta
     4681 gtttggctcc tgcccgtttc ggcgtccact tgcacaaaca caaacaagca ttatatataa
     4741 ggctcgtctc tccctcccaa ccacactcac ttttttgccc gtcttccctt gctaacacaa
     4801 aagtcaagaa cacaaacaac caccccaacc cccttacaca caagacatat ctacagcaat
     4861 gggtaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     4921 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     4981 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     5041 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     5101 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     5161 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggcaatgga
     5221 tgccattgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     5281 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     5341 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     5401 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     5461 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     5521 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     5581 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     5641 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     5701 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     5761 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
     5821 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     5881 ggaataatca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct
     5941 ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata
     6001 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga
     6061 cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg
     6121 agcaggcttg gaggcgacgt ggcagtggct cgcgggagga tcggcgggaa actccgggat
     6181 atccgtcgag agtttacacg tgaatgggca gcgcaatccg ttgacgacga tacgactggc
     6241 aaagtagcga cgatacctgc cagacaggtg acatgtgcag gccgcactaa caaggaaacg
     6301 ggcgctgggg ggggcgggct tctagacttt gcccttgaac aggaatctag tgggggcttg
     6361 tctttccgcc aatgggggag cgcctgttga gcgaccgtgc atgctggaac gccaagtgta
     6421 tgtacagctg gtggtctcgc agcggtatgt gacgggactt acatctctcg ttttttcatg
     6481 accacgtttt cacaggctcg gaggtacgtt aaagttttga aggctgcatc tgaaccgagg
     6541 tatgggggag tttgaacagc aacagtgttg gggctgaggg ggccaagatc ggggcaagca
     6601 gaggtcttag atcaattgtg gggatcccaa agggctcgtt atcacctttt tccacccaat
     6661 tcgggtccca attgatccac tactggcttg cccaagttac cccagaaatg ccgccccgga
     6721 tttctccaaa aacctaataa gcttcatgga acttggtgga agtgactttc tacagagtgg
     6781 agagaaccgt ggacacgtgg caatggcgct gaccgtgtcc ccgagccgaa tcgacgtgag
     6841 gggagaacgg agtatctgcg gtcatgtgac cttccagagc ggcgtcgcca gtgtgcacgc
     6901 ggtgaccccc agtttgggtc tctgtcacac gcatactacc tcggctctcc acatgctgaa
     6961 ctttatcttt cgtggggatc ataccgaaag ttgcaactac caggtgtata taaagcctgg
     7021 tagactcccc ccactttgga cctcatccaa ccaagacaca caaaaatgga ccgacttaac
     7081 aacctcgcca cccagctcga gcagaacccc gccaa
//
