LOCUS       sequence-435425-        7113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The base plasmid of TUNEYALI for TF34.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7113)
  TITLE     Regulating transcription factors in Yarrowia lipolytica
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7113)
  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            "Unpublished"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: pCfb13224
FEATURES             Location/Qualifiers
     source          1..7113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        364..438
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(843..876)
                     /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(1083..1102)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1108..1127)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1169..1260
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1261..1329,1330..2121)
                     /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(1479..1498)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2243..2831
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2732..2751
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2985..3002
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3100..3117
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3101..3117
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3587..3620
                     /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             4762..5790
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4762..5790
                     /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(5807..5825)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 tgaggcatta aatagagctt gatatataaa atgttacatt tcacagtctg aacttttgca
       61 gattacctaa tttggtaaga tattaattat gaactgaaag ttgatggcat ccctaaattt
      121 gatgcaacgg aatgcgtgcg atagtgaatc attgctaaca gatcaagtaa tcgtatcaat
      181 cgttccacgt cttatccaga ctcgatttcc gccctaacct aaggaaactg ctgagtaatt
      241 tcgaggttgg ttcctaacaa aagccaagaa acgcgccgat ggtttagtgg taaaatccat
      301 cgttgccatc gatgggcccc cggttcgatt ccgggtcggc gcactcaaca taaagtttta
      361 ccggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
      421 agtggcaccg agtcggtggt gctttttttg tgtgggtacg gtgcaggtgc caactggagc
      481 gatcgaattc gtaacaaatt catcgctgca attattgtag ctatcgaagg gaacaaccgg
      541 tataatagga aactgataat atcaaaatac aggaaattgc ccggaaatta cgaacgtgtt
      601 aaaatatcat caaaaaatct gcaagactga agagctcttc aatgatatct caaattacca
      661 ccagcagtat cacaaacacc acacacatca tgtcggacat tgaagataag aaggtggaga
      721 gtctggactt cgacccctct ctcaagaaga agaagaagaa gtccaagcct gcatttgtgg
      781 aggatgacga ggtctccgag gttatcgcgt gcattcgcgg ccgcatttaa atccccactt
      841 cagaagttcc tatactttct agagaatagg aacttcacta tagagtcgaa taagggcgac
      901 accccataat tagcccgggc gaaaggccca gtctttcgac tgagcctttc gttttatttg
      961 atgcctggca gttccctact ctcgcatggg gagtccccac actaccatcg gcgctacggc
     1021 gtttcacttc tgagttcggc atggggtcag gtgggaccac cgcgctactg ccgccaggca
     1081 aacaaggggt gttatgagcc atattcaggt ataaatgggc tcgcgataat gttcagaatt
     1141 ggttaattgg ttgtaacact gacccctatt tgtttatttt tctaaataca ttcaaatatg
     1201 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagaat
     1261 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1321 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     1381 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     1441 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     1501 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     1561 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     1621 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     1681 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     1741 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     1801 cctgtagcga tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     1861 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     1921 tcggcccttc cggctggctg gtttattgct gataaatccg gagccggtga gcgtggttct
     1981 cgcggtatca tcgcagcgct ggggccagat ggtaagccct cccgtatcgt agttatctac
     2041 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2101 tcactgatta agcattggta agcagagcat tacgctgact tgacgggacg gcgcaagctc
     2161 atgaccaaaa tcccttaacg tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg
     2221 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2281 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2341 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     2401 agccgtagtt agcccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2461 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2521 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2581 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2641 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2701 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2761 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2821 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2881 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2941 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3001 aggaagcgga aggcgagagt agggaactgc caggcatcaa actaagcaga aggcccctga
     3061 cggatggcct ttttgcgttt ctacaaactc tttctgtgtt gtaaaacgac ggccagtctt
     3121 aagctcgggc cccctgggcg gttctgataa cgagtaatcg ttaatccgca aataacgtaa
     3181 aaacccgctt cggcgggttt ttttatgggg ggagtttagg gaaagagcat ttgtcagaat
     3241 atttaagggc gcctgtcact ttgcttgata tatgagaatt atttaacctt ataaatgaga
     3301 aaaaagcaac gcactttaaa taagatacgt tgctttttcg attgatgaac acctataatt
     3361 aaactattca tctattattt atgatttttt gtatatacaa tatttctagt ttgttaaaga
     3421 gaattaagaa aataaatctc gaaaataata aagggaaaat cagtttttga tatcaaaatt
     3481 atacatgtca acgataatac aaaatataat acaaactata agatgttatc agtatttatt
     3541 atgcatttag aataaatttt gtgtcgccct tattcgactc actatagaag ttcctattct
     3601 ctagaaagta taggaacttc acttcatttt atttaaattt gcggccgcta tgtctgataa
     3661 aaggatgtaa cataggcaag ctgctcgtga gtgttgagta cgaaccttag atccaaatca
     3721 cccgcaccca cggatatact tgcttgaata tacagtagta aggagtttgg cgcccgtttt
     3781 ttcgagcccc acacgtttcg gtgagtatga gcggcggcag attcgagcgt ttccggtttc
     3841 cgcggctgga cgagagccca tgatgggggc tcccaccacc agcaatcagg gccctgatta
     3901 cacacccacc tgtaatgtca tgctgttcat cgtggttaat gctgctgtgt gctgtgtgtg
     3961 tgtgttgttt ggcgctcatt gttgcgttat gcagcgtaca ccacaatatt ggaagcttat
     4021 tagcctttct attttttcgt ttgcaaggct taacaacatt gctgtggaga gggatgggga
     4081 tatggaggcc gctggaggga gtcggagagg cgttttggag cggcttggcc tggcgcccag
     4141 ctcgcgaaac gcacctagga ccctttggca cgccgaaatg tgccactttt cagtctagta
     4201 acgccttacc tacgtcattc catgcatgca tgtttgcgcc ttttttccct tgcccttgat
     4261 cgccacacag tacagtgcac tgtacagtgg aggttttggg ggggtcttag atgggagcta
     4321 aaagcggcct agcggtacac tagtgggatt gtatggagtg gcatggagcc taggtggagc
     4381 ctgacaggac gcacgaccgg ctagcccgtg acagacgatg ggtggctcct gttgtccacc
     4441 gcgtacaaat gtttgggcca aagtcttgtc agccttgctt gcgaacctaa ttcccaattt
     4501 tgtcacttcg cacccccatt gatcgagccc taacccctgc ccatcaggca atccaattaa
     4561 gctcgcattg tctgccttgt ttagtttggc tcctgcccgt ttcggcgtcc acttgcacaa
     4621 acacaaacaa gcattatata taaggctcgt ctctccctcc caaccacact cacttttttg
     4681 cccgtcttcc cttgctaaca caaaagtcaa gaacacaaac aaccacccca acccccttac
     4741 acacaagaca tatctacagc aatgggtaaa aagcctgaac tcaccgcgac gtctgtcgag
     4801 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     4861 gaatctcgtg ctttcagctt cgatgtmgga gggcgtggat atgtcctgcg ggtaaatagc
     4921 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     4981 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     5041 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     5101 cagccggtcg cggaggcaat ggatgccatt gctgcggccg atcttagcca gacgagcggg
     5161 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     5221 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     5281 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     5341 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     5401 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     5461 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     5521 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     5581 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     5641 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     5701 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     5761 cccagcactc gtccgagggc aaaggaataa tcatgtaatt agttatgtca cgcttacatt
     5821 cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct
     5881 aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa
     5941 tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc
     6001 ttgagaaggt tttgggacgc tcgagcaggc ttggaggcga cgtggcagtg gctcgcggga
     6061 ggatcggcgg gaaactccgg gatatccgtc gagagtttac acgtgaatgg gcagcgcaat
     6121 ccgttgacga cgatacgact ggcaaagtag cgacgatacc tgccagacag gtgacatgtg
     6181 caggccgcac taacaaggaa acgggcgctg gggggggcgg gcttctagac tttgcccttg
     6241 aacaggaatc tagtgggggc ttgtctttcc gccaatgggg gagcgcctgt tgagcgaccg
     6301 tgcatgctgg aacgccaagt gtatgtacag ctggtggtct cgcagcggta tgtgacggga
     6361 cttacatctc tcgttttttc atgaccacgt tttcacaggc tcggaggtac gttaaagttt
     6421 tgaaggctgc atctgaaccg aggtatgggg gagtttgaac agcaacagtg ttggggctga
     6481 gggggccaag atcggggcaa gcagaggtct tagatcaatt gtggggatcc caaagggctc
     6541 gttatcacct ttttccaccc aattcgggtc ccaattgatc cactactggc ttgcccaagt
     6601 taccccagaa atgccgcccc ggatttctcc aaaaacctaa taagcttcat ggaacttggt
     6661 ggaagtgact ttctacagag tggagagaac cgtggacacg tggcaatggc gctgaccgtg
     6721 tccccgagcc gaatcgacgt gaggggagaa cggagtatct gcggtcatgt gaccttccag
     6781 agcggcgtcg ccagtgtgca cgcggtgacc cccagtttgg gtctctgtca cacgcatact
     6841 acctcggctc tccacatgct gaactttatc tttcgtgggg atcataccga aagttgcaac
     6901 taccaggtgt atataaagcc tggtagactc cccccacttt ggacctcatc caaccaagac
     6961 acacaaaaat ggaccgactt aacaacctcg ccacccagct cgagcagaac cccgccaatt
     7021 cgtagataat ggaatacaaa tggatatcca gagtatacac atggatagta tacactgaca
     7081 cgacaattct gtatctcttt atgttaacta ctg
//
