LOCUS       sequence-435394-        7113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The base plasmid of TUNEYALI for TF25.
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  JOURNAL   Unpublished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            "Unpublished"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: pCfb13215
FEATURES             Location/Qualifiers
     source          1..7113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             520..1548
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             520..1548
                     /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(1565..1583)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     misc_RNA        3235..3309
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3714..3747)
                     /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(3954..3973)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3979..3998)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4040..4131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4132..4200,4201..4992)
                     /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(4350..4369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5114..5702
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5603..5622
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5856..5873
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5971..5988
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5972..5988
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6458..6491
                     /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)"
ORIGIN
        1 ttttcccttg cccttgatcg ccacacagta cagtgcactg tacagtggag gttttggggg
       61 ggtcttagat gggagctaaa agcggcctag cggtacacta gtgggattgt atggagtggc
      121 atggagccta ggtggagcct gacaggacgc acgaccggct agcccgtgac agacgatggg
      181 tggctcctgt tgtccaccgc gtacaaatgt ttgggccaaa gtcttgtcag ccttgcttgc
      241 gaacctaatt cccaattttg tcacttcgca cccccattga tcgagcccta acccctgccc
      301 atcaggcaat ccaattaagc tcgcattgtc tgccttgttt agtttggctc ctgcccgttt
      361 cggcgtccac ttgcacaaac acaaacaagc attatatata aggctcgtct ctccctccca
      421 accacactca cttttttgcc cgtcttccct tgctaacaca aaagtcaaga acacaaacaa
      481 ccaccccaac ccccttacac acaagacata tctacagcaa tgggtaaaaa gcctgaactc
      541 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg
      601 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat
      661 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac
      721 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc
      781 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc
      841 gaactgcccg ctgttctgca gccggtcgcg gaggcaatgg atgccattgc tgcggccgat
      901 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca
      961 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg
     1021 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag
     1081 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg
     1141 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa
     1201 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg
     1261 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg
     1321 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca
     1381 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt
     1441 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc
     1501 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaataatc atgtaattag
     1561 ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag
     1621 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     1681 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
     1741 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gagcaggctt ggaggcgacg
     1801 tggcagtggc tcgcgggagg atcggcggga aactccggga tatccgtcga gagtttacac
     1861 gtgaatgggc agcgcaatcc gttgacgacg atacgactgg caaagtagcg acgatacctg
     1921 ccagacaggt gacatgtgca ggccgcacta acaaggaaac gggcgctggg gggggcgggc
     1981 ttctagactt tgcccttgaa caggaatcta gtgggggctt gtctttccgc caatggggga
     2041 gcgcctgttg agcgaccgtg catgctggaa cgccaagtgt atgtacagct ggtggtctcg
     2101 cagcggtatg tgacgggact tacatctctc gttttttcat gaccacgttt tcacaggctc
     2161 ggaggtacgt taaagttttg aaggctgcat ctgaaccgag gtatggggga gtttgaacag
     2221 caacagtgtt ggggctgagg gggccaagat cggggcaagc agaggtctta gatcaattgt
     2281 ggggatccca aagggctcgt tatcaccttt ttccacccaa ttcgggtccc aattgatcca
     2341 ctactggctt gcccaagtta ccccagaaat gccgccccgg atttctccaa aaacctaata
     2401 agcttcatgg aacttggtgg aagtgacttt ctacagagtg gagagaaccg tggacacgtg
     2461 gcaatggcgc tgaccgtgtc cccgagccga atcgacgtga ggggagaacg gagtatctgc
     2521 ggtcatgtga ccttccagag cggcgtcgcc agtgtgcacg cggtgacccc cagtttgggt
     2581 ctctgtcaca cgcatactac ctcggctctc cacatgctga actttatctt tcgtggggat
     2641 cataccgaaa gttgcaacta ccaggtgtat ataaagcctg gtagactccc cccactttgg
     2701 acctcatcca accaagacac acaaaaatgg accgacttaa caacctcgcc acccagctcg
     2761 agcagaaccc cgccaattcg tagataatgg aatacaaatg gatatccaga gtatacacat
     2821 ggatagtata cactgacacg acaattctgt atctctttat gttaactact gtgaggcatt
     2881 aaatagagct tgatatataa aatgttacat ttcacagtct gaacttttgc agattaccta
     2941 atttggtaag atattaatta tgaactgaaa gttgatggca tccctaaatt tgatgcaacg
     3001 gaatgcgtgc gatagtgaat cattgctaac agatcaagta atcgtatcaa tcgttccacg
     3061 tcttatccag actcgatttc cgccctaacc taaggaaact gctgagtaat ttcgaggttg
     3121 gttcctaaca aaagccaaga aacgcgccga tggtttagtg gtaaaatcca tcgttgccat
     3181 cgatgggccc ccggttcgat tccgggtcgg cgcaagtagt cagagggaga aatggtttta
     3241 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     3301 gagtcggtgg tgcttttttt gtgtgggtac ggtacaacct taaatccaag gttaaactcg
     3361 ccgtccgggt aacttttaga ctttgatcat tccttcactt ttagagtgag ttgggaaatg
     3421 gcgctgtatt tccgagatgg cagagttcat gggaccagta gttcacgata acgatcgtga
     3481 aaaaaatggg agtcggactg aagagctctt caatgaatgt gggcggtgac tctgtcgaca
     3541 tgttgctatc ttccgtctcg gcccaccacc ggtcctctga cgcgggtcag agcgatatgg
     3601 gctccatctc cccctccacc gcacacacta cccccgacgc caccacttac aagacgtcag
     3661 acgaggagga cgccatcgcg tgcattcgcg gccgcattta aatccccact tcagaagttc
     3721 ctatactttc tagagaatag gaacttcact atagagtcga ataagggcga caccccataa
     3781 ttagcccggg cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgcctggc
     3841 agttccctac tctcgcatgg ggagtcccca cactaccatc ggcgctacgg cgtttcactt
     3901 ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc aaacaagggg
     3961 tgttatgagc catattcagg tataaatggg ctcgcgataa tgttcagaat tggttaattg
     4021 gttgtaacac tgacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4081 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagaa tatgagtatt
     4141 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4201 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4261 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4321 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4381 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4441 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4501 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4561 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4621 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagcg
     4681 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4741 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4801 ccggctggct ggtttattgc tgataaatcc ggagccggtg agcgtggttc tcgcggtatc
     4861 atcgcagcgc tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4921 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4981 aagcattggt aagcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa
     5041 atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga
     5101 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5161 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5221 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5281 tagcccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5341 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5401 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5461 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5521 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5581 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5641 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5701 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5761 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     5821 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     5881 aaggcgagag tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc
     5941 tttttgcgtt tctacaaact ctttctgtgt tgtaaaacga cggccagtct taagctcggg
     6001 ccccctgggc ggttctgata acgagtaatc gttaatccgc aaataacgta aaaacccgct
     6061 tcggcgggtt tttttatggg gggagtttag ggaaagagca tttgtcagaa tatttaaggg
     6121 cgcctgtcac tttgcttgat atatgagaat tatttaacct tataaatgag aaaaaagcaa
     6181 cgcactttaa ataagatacg ttgctttttc gattgatgaa cacctataat taaactattc
     6241 atctattatt tatgattttt tgtatataca atatttctag tttgttaaag agaattaaga
     6301 aaataaatct cgaaaataat aaagggaaaa tcagtttttg atatcaaaat tatacatgtc
     6361 aacgataata caaaatataa tacaaactat aagatgttat cagtatttat tatgcattta
     6421 gaataaattt tgtgtcgccc ttattcgact cactatagaa gttcctattc tctagaaagt
     6481 ataggaactt cacttcattt tatttaaatt tgcggccgct atgtctgata aaaggatgta
     6541 acataggcaa gctgctcgtg agtgttgagt acgaacctta gatccaaatc acccgcaccc
     6601 acggatatac ttgcttgaat atacagtagt aaggagtttg gcgcccgttt tttcgagccc
     6661 cacacgtttc ggtgagtatg agcggcggca gattcgagcg tttccggttt ccgcggctgg
     6721 acgagagccc atgatggggg ctcccaccac cagcaatcag ggccctgatt acacacccac
     6781 ctgtaatgtc atgctgttca tcgtggttaa tgctgctgtg tgctgtgtgt gtgtgttgtt
     6841 tggcgctcat tgttgcgtta tgcagcgtac accacaatat tggaagctta ttagcctttc
     6901 tattttttcg tttgcaaggc ttaacaacat tgctgtggag agggatgggg atatggaggc
     6961 cgctggaggg agtcggagag gcgttttgga gcggcttggc ctggcgccca gctcgcgaaa
     7021 cgcacctagg accctttggc acgccgaaat gtgccacttt tcagtctagt aacgccttac
     7081 ctacgtcatt ccatgcatgc atgtttgcgc ctt
//
