LOCUS       sequence-435424-        7113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The base plasmid of TUNEYALI for TF24.
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  JOURNAL   Unpublished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            "Unpublished"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: pCfb13214
FEATURES             Location/Qualifiers
     source          1..7113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        282..356
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(761..794)
                     /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(1001..1020)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1026..1045)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1087..1178
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1179..1247,1248..2039)
                     /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(1397..1416)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2161..2749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2650..2669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2903..2920
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3018..3035
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3019..3035
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3505..3538
                     /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             4680..5708
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4680..5708
                     /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(5725..5743)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 ttaattatga actgaaagtt gatggcatcc ctaaatttga tgcaacggaa tgcgtgcgat
       61 agtgaatcat tgctaacaga tcaagtaatc gtatcaatcg ttccacgtct tatccagact
      121 cgatttccgc cctaacctaa ggaaactgct gagtaatttc gaggttggtt cctaacaaaa
      181 gccaagaaac gcgccgatgg tttagtggta aaatccatcg ttgccatcga tgggcccccg
      241 gttcgattcc gggtcggcgc aatgggagtg tgtgatagac ggttttagag ctagaaatag
      301 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtggtgc
      361 tttttttgtg tgggtacggt aaggaagaga aaaatacaga ggccacagga gacactatat
      421 taataagtgg tgtcgcctag ggtcaggtga tgcgctagtg caaatatacg aggctcgacg
      481 agaattcttc acatcaccga tactttgctc ttgtcaagtc accaaagctc ctcgacacaa
      541 tagactgaag agctcttcaa tgatggccta tacagaagtc aagtcggaac tgcccatata
      601 cgggggacac ccacaaaacc caacaagccc acattttgac accgtggaag actgttgtct
      661 tctgcgcttc tcgcgtctgt gtcaaacgac gccacaggca ctcgacttct tgtgtcaaaa
      721 catcgcgtgc attcgcggcc gcatttaaat ccccacttca gaagttccta tactttctag
      781 agaataggaa cttcactata gagtcgaata agggcgacac cccataatta gcccgggcga
      841 aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctggcagt tccctactct
      901 cgcatgggga gtccccacac taccatcggc gctacggcgt ttcacttctg agttcggcat
      961 ggggtcaggt gggaccaccg cgctactgcc gccaggcaaa caaggggtgt tatgagccat
     1021 attcaggtat aaatgggctc gcgataatgt tcagaattgg ttaattggtt gtaacactga
     1081 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1141 cctgataaat gcttcaataa tattgaaaaa ggaagaatat gagtattcaa catttccgtg
     1201 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1261 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1321 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1381 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1441 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1501 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1561 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1621 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1681 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcgatg gcaacaacgt
     1741 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1801 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1861 ttattgctga taaatccgga gccggtgagc gtggttctcg cggtatcatc gcagcgctgg
     1921 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1981 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaag
     2041 cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg
     2101 agttacgcgc gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2161 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2221 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2281 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag cccaccactt
     2341 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2401 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2461 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2521 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2581 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2641 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2701 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2761 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2821 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     2881 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag gcgagagtag
     2941 ggaactgcca ggcatcaaac taagcagaag gcccctgacg gatggccttt ttgcgtttct
     3001 acaaactctt tctgtgttgt aaaacgacgg ccagtcttaa gctcgggccc cctgggcggt
     3061 tctgataacg agtaatcgtt aatccgcaaa taacgtaaaa acccgcttcg gcgggttttt
     3121 ttatgggggg agtttaggga aagagcattt gtcagaatat ttaagggcgc ctgtcacttt
     3181 gcttgatata tgagaattat ttaaccttat aaatgagaaa aaagcaacgc actttaaata
     3241 agatacgttg ctttttcgat tgatgaacac ctataattaa actattcatc tattatttat
     3301 gattttttgt atatacaata tttctagttt gttaaagaga attaagaaaa taaatctcga
     3361 aaataataaa gggaaaatca gtttttgata tcaaaattat acatgtcaac gataatacaa
     3421 aatataatac aaactataag atgttatcag tatttattat gcatttagaa taaattttgt
     3481 gtcgccctta ttcgactcac tatagaagtt cctattctct agaaagtata ggaacttcac
     3541 ttcattttat ttaaatttgc ggccgctatg tctgataaaa ggatgtaaca taggcaagct
     3601 gctcgtgagt gttgagtacg aaccttagat ccaaatcacc cgcacccacg gatatacttg
     3661 cttgaatata cagtagtaag gagtttggcg cccgtttttt cgagccccac acgtttcggt
     3721 gagtatgagc ggcggcagat tcgagcgttt ccggtttccg cggctggacg agagcccatg
     3781 atgggggctc ccaccaccag caatcagggc cctgattaca cacccacctg taatgtcatg
     3841 ctgttcatcg tggttaatgc tgctgtgtgc tgtgtgtgtg tgttgtttgg cgctcattgt
     3901 tgcgttatgc agcgtacacc acaatattgg aagcttatta gcctttctat tttttcgttt
     3961 gcaaggctta acaacattgc tgtggagagg gatggggata tggaggccgc tggagggagt
     4021 cggagaggcg ttttggagcg gcttggcctg gcgcccagct cgcgaaacgc acctaggacc
     4081 ctttggcacg ccgaaatgtg ccacttttca gtctagtaac gccttaccta cgtcattcca
     4141 tgcatgcatg tttgcgcctt ttttcccttg cccttgatcg ccacacagta cagtgcactg
     4201 tacagtggag gttttggggg ggtcttagat gggagctaaa agcggcctag cggtacacta
     4261 gtgggattgt atggagtggc atggagccta ggtggagcct gacaggacgc acgaccggct
     4321 agcccgtgac agacgatggg tggctcctgt tgtccaccgc gtacaaatgt ttgggccaaa
     4381 gtcttgtcag ccttgcttgc gaacctaatt cccaattttg tcacttcgca cccccattga
     4441 tcgagcccta acccctgccc atcaggcaat ccaattaagc tcgcattgtc tgccttgttt
     4501 agtttggctc ctgcccgttt cggcgtccac ttgcacaaac acaaacaagc attatatata
     4561 aggctcgtct ctccctccca accacactca cttttttgcc cgtcttccct tgctaacaca
     4621 aaagtcaaga acacaaacaa ccaccccaac ccccttacac acaagacata tctacagcaa
     4681 tgggtaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     4741 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     4801 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     4861 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     4921 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     4981 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggcaatgg
     5041 atgccattgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     5101 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     5161 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     5221 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     5281 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     5341 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     5401 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     5461 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     5521 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     5581 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     5641 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     5701 aggaataatc atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc
     5761 tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat
     5821 agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag
     5881 acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc
     5941 gagcaggctt ggaggcgacg tggcagtggc tcgcgggagg atcggcggga aactccggga
     6001 tatccgtcga gagtttacac gtgaatgggc agcgcaatcc gttgacgacg atacgactgg
     6061 caaagtagcg acgatacctg ccagacaggt gacatgtgca ggccgcacta acaaggaaac
     6121 gggcgctggg gggggcgggc ttctagactt tgcccttgaa caggaatcta gtgggggctt
     6181 gtctttccgc caatggggga gcgcctgttg agcgaccgtg catgctggaa cgccaagtgt
     6241 atgtacagct ggtggtctcg cagcggtatg tgacgggact tacatctctc gttttttcat
     6301 gaccacgttt tcacaggctc ggaggtacgt taaagttttg aaggctgcat ctgaaccgag
     6361 gtatggggga gtttgaacag caacagtgtt ggggctgagg gggccaagat cggggcaagc
     6421 agaggtctta gatcaattgt ggggatccca aagggctcgt tatcaccttt ttccacccaa
     6481 ttcgggtccc aattgatcca ctactggctt gcccaagtta ccccagaaat gccgccccgg
     6541 atttctccaa aaacctaata agcttcatgg aacttggtgg aagtgacttt ctacagagtg
     6601 gagagaaccg tggacacgtg gcaatggcgc tgaccgtgtc cccgagccga atcgacgtga
     6661 ggggagaacg gagtatctgc ggtcatgtga ccttccagag cggcgtcgcc agtgtgcacg
     6721 cggtgacccc cagtttgggt ctctgtcaca cgcatactac ctcggctctc cacatgctga
     6781 actttatctt tcgtggggat cataccgaaa gttgcaacta ccaggtgtat ataaagcctg
     6841 gtagactccc cccactttgg acctcatcca accaagacac acaaaaatgg accgacttaa
     6901 caacctcgcc acccagctcg agcagaaccc cgccaattcg tagataatgg aatacaaatg
     6961 gatatccaga gtatacacat ggatagtata cactgacacg acaattctgt atctctttat
     7021 gttaactact gtgaggcatt aaatagagct tgatatataa aatgttacat ttcacagtct
     7081 gaacttttgc agattaccta atttggtaag ata
//
