LOCUS       sequence-436035-        7113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The base plasmid of TUNEYALI for TF14.
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  JOURNAL   Unpublished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            "Unpublished"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: pCfb13204
FEATURES             Location/Qualifiers
     source          1..7113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        322..396
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(801..834)
                     /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(1041..1060)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1066..1085)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1127..1218
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1219..1287,1288..2079)
                     /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(1437..1456)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2201..2789
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2690..2709
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2943..2960
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3058..3075
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3059..3075
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3545..3578
                     /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             4720..5748
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4720..5748
                     /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(5765..5783)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 acagtctgaa cttttgcaga ttacctaatt tggtaagata ttaattatga actgaaagtt
       61 gatggcatcc ctaaatttga tgcaacggaa tgcgtgcgat agtgaatcat tgctaacaga
      121 tcaagtaatc gtatcaatcg ttccacgtct tatccagact cgatttccgc cctaacctaa
      181 ggaaactgct gagtaatttc gaggttggtt cctaacaaaa gccaagaaac gcgccgatgg
      241 tttagtggta aaatccatcg ttgccatcga tgggcccccg gttcgattcc gggtcggcgc
      301 agtgtagtgt ggcgtgtacg ggttttagag ctagaaatag caagttaaaa taaggctagt
      361 ccgttatcaa cttgaaaaag tggcaccgag tcggtggtgc tttttttgtg tgggtacggt
      421 gggcgtcgaa tccccctttc ttctcttttg agaagcggtc tttttccttt cattcttggt
      481 ttcgtccaag tcaccccccg cacctgtttt ggcctggtgt ttgagaaaga ccgccgagga
      541 cagcgaaaac agccggtcgt cttcgttgaa cacgtagcgc ttgactgaag agctcttcaa
      601 tgtcgttccg gcaacagttc cagcagggag gcaatcccaa tggacagatg ggcttcaaca
      661 acatgaacat gagcccaaac atgaacaacg cgaaaatcgg aggcatggat atgggagcgc
      721 agatgggcca gatgggccag atgggccaga tgccacagat gatcgcgtgc attcgcggcc
      781 gcatttaaat ccccacttca gaagttccta tactttctag agaataggaa cttcactata
      841 gagtcgaata agggcgacac cccataatta gcccgggcga aaggcccagt ctttcgactg
      901 agcctttcgt tttatttgat gcctggcagt tccctactct cgcatgggga gtccccacac
      961 taccatcggc gctacggcgt ttcacttctg agttcggcat ggggtcaggt gggaccaccg
     1021 cgctactgcc gccaggcaaa caaggggtgt tatgagccat attcaggtat aaatgggctc
     1081 gcgataatgt tcagaattgg ttaattggtt gtaacactga cccctatttg tttatttttc
     1141 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     1201 tattgaaaaa ggaagaatat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1261 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1321 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1381 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     1441 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     1501 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     1561 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     1621 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     1681 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     1741 gagcgtgaca ccacgatgcc tgtagcgatg gcaacaacgt tgcgcaaact attaactggc
     1801 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     1861 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatccgga
     1921 gccggtgagc gtggttctcg cggtatcatc gcagcgctgg ggccagatgg taagccctcc
     1981 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     2041 atcgctgaga taggtgcctc actgattaag cattggtaag cagagcatta cgctgacttg
     2101 acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgc gcgtcgttcc
     2161 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2221 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2281 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2341 atactgttct tctagtgtag ccgtagttag cccaccactt caagaactct gtagcaccgc
     2401 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2461 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2521 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2581 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2641 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2701 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2761 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2821 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     2881 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     2941 gcagcgagtc agtgagcgag gaagcggaag gcgagagtag ggaactgcca ggcatcaaac
     3001 taagcagaag gcccctgacg gatggccttt ttgcgtttct acaaactctt tctgtgttgt
     3061 aaaacgacgg ccagtcttaa gctcgggccc cctgggcggt tctgataacg agtaatcgtt
     3121 aatccgcaaa taacgtaaaa acccgcttcg gcgggttttt ttatgggggg agtttaggga
     3181 aagagcattt gtcagaatat ttaagggcgc ctgtcacttt gcttgatata tgagaattat
     3241 ttaaccttat aaatgagaaa aaagcaacgc actttaaata agatacgttg ctttttcgat
     3301 tgatgaacac ctataattaa actattcatc tattatttat gattttttgt atatacaata
     3361 tttctagttt gttaaagaga attaagaaaa taaatctcga aaataataaa gggaaaatca
     3421 gtttttgata tcaaaattat acatgtcaac gataatacaa aatataatac aaactataag
     3481 atgttatcag tatttattat gcatttagaa taaattttgt gtcgccctta ttcgactcac
     3541 tatagaagtt cctattctct agaaagtata ggaacttcac ttcattttat ttaaatttgc
     3601 ggccgctatg tctgataaaa ggatgtaaca taggcaagct gctcgtgagt gttgagtacg
     3661 aaccttagat ccaaatcacc cgcacccacg gatatacttg cttgaatata cagtagtaag
     3721 gagtttggcg cccgtttttt cgagccccac acgtttcggt gagtatgagc ggcggcagat
     3781 tcgagcgttt ccggtttccg cggctggacg agagcccatg atgggggctc ccaccaccag
     3841 caatcagggc cctgattaca cacccacctg taatgtcatg ctgttcatcg tggttaatgc
     3901 tgctgtgtgc tgtgtgtgtg tgttgtttgg cgctcattgt tgcgttatgc agcgtacacc
     3961 acaatattgg aagcttatta gcctttctat tttttcgttt gcaaggctta acaacattgc
     4021 tgtggagagg gatggggata tggaggccgc tggagggagt cggagaggcg ttttggagcg
     4081 gcttggcctg gcgcccagct cgcgaaacgc acctaggacc ctttggcacg ccgaaatgtg
     4141 ccacttttca gtctagtaac gccttaccta cgtcattcca tgcatgcatg tttgcgcctt
     4201 ttttcccttg cccttgatcg ccacacagta cagtgcactg tacagtggag gttttggggg
     4261 ggtcttagat gggagctaaa agcggcctag cggtacacta gtgggattgt atggagtggc
     4321 atggagccta ggtggagcct gacaggacgc acgaccggct agcccgtgac agacgatggg
     4381 tggctcctgt tgtccaccgc gtacaaatgt ttgggccaaa gtcttgtcag ccttgcttgc
     4441 gaacctaatt cccaattttg tcacttcgca cccccattga tcgagcccta acccctgccc
     4501 atcaggcaat ccaattaagc tcgcattgtc tgccttgttt agtttggctc ctgcccgttt
     4561 cggcgtccac ttgcacaaac acaaacaagc attatatata aggctcgtct ctccctccca
     4621 accacactca cttttttgcc cgtcttccct tgctaacaca aaagtcaaga acacaaacaa
     4681 ccaccccaac ccccttacac acaagacata tctacagcaa tgggtaaaaa gcctgaactc
     4741 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg
     4801 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat
     4861 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac
     4921 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc
     4981 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc
     5041 gaactgcccg ctgttctgca gccggtcgcg gaggcaatgg atgccattgc tgcggccgat
     5101 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca
     5161 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg
     5221 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag
     5281 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg
     5341 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa
     5401 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg
     5461 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg
     5521 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca
     5581 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt
     5641 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc
     5701 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaataatc atgtaattag
     5761 ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag
     5821 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     5881 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
     5941 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gagcaggctt ggaggcgacg
     6001 tggcagtggc tcgcgggagg atcggcggga aactccggga tatccgtcga gagtttacac
     6061 gtgaatgggc agcgcaatcc gttgacgacg atacgactgg caaagtagcg acgatacctg
     6121 ccagacaggt gacatgtgca ggccgcacta acaaggaaac gggcgctggg gggggcgggc
     6181 ttctagactt tgcccttgaa caggaatcta gtgggggctt gtctttccgc caatggggga
     6241 gcgcctgttg agcgaccgtg catgctggaa cgccaagtgt atgtacagct ggtggtctcg
     6301 cagcggtatg tgacgggact tacatctctc gttttttcat gaccacgttt tcacaggctc
     6361 ggaggtacgt taaagttttg aaggctgcat ctgaaccgag gtatggggga gtttgaacag
     6421 caacagtgtt ggggctgagg gggccaagat cggggcaagc agaggtctta gatcaattgt
     6481 ggggatccca aagggctcgt tatcaccttt ttccacccaa ttcgggtccc aattgatcca
     6541 ctactggctt gcccaagtta ccccagaaat gccgccccgg atttctccaa aaacctaata
     6601 agcttcatgg aacttggtgg aagtgacttt ctacagagtg gagagaaccg tggacacgtg
     6661 gcaatggcgc tgaccgtgtc cccgagccga atcgacgtga ggggagaacg gagtatctgc
     6721 ggtcatgtga ccttccagag cggcgtcgcc agtgtgcacg cggtgacccc cagtttgggt
     6781 ctctgtcaca cgcatactac ctcggctctc cacatgctga actttatctt tcgtggggat
     6841 cataccgaaa gttgcaacta ccaggtgtat ataaagcctg gtagactccc cccactttgg
     6901 acctcatcca accaagacac acaaaaatgg accgacttaa caacctcgcc acccagctcg
     6961 agcagaaccc cgccaattcg tagataatgg aatacaaatg gatatccaga gtatacacat
     7021 ggatagtata cactgacacg acaattctgt atctctttat gttaactact gtgaggcatt
     7081 aaatagagct tgatatataa aatgttacat ttc
//
