LOCUS       sequence-435519-        7113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The base plasmid of TUNEYALI for TF27.
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  JOURNAL   Unpublished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            "Unpublished"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: pCfb13217
FEATURES             Location/Qualifiers
     source          1..7113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             738..1766
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             738..1766
                     /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(1783..1801)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     misc_RNA        3453..3527
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3932..3965)
                     /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(4172..4191)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4197..4216)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4258..4349
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4350..4418,4419..5210)
                     /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(4568..4587)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5332..5920
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5821..5840
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6074..6091
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6189..6206
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6190..6206
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6676..6709
                     /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 ctttctattt tttcgtttgc aaggcttaac aacattgctg tggagaggga tggggatatg
       61 gaggccgctg gagggagtcg gagaggcgtt ttggagcggc ttggcctggc gcccagctcg
      121 cgaaacgcac ctaggaccct ttggcacgcc gaaatgtgcc acttttcagt ctagtaacgc
      181 cttacctacg tcattccatg catgcatgtt tgcgcctttt ttcccttgcc cttgatcgcc
      241 acacagtaca gtgcactgta cagtggaggt tttggggggg tcttagatgg gagctaaaag
      301 cggcctagcg gtacactagt gggattgtat ggagtggcat ggagcctagg tggagcctga
      361 caggacgcac gaccggctag cccgtgacag acgatgggtg gctcctgttg tccaccgcgt
      421 acaaatgttt gggccaaagt cttgtcagcc ttgcttgcga acctaattcc caattttgtc
      481 acttcgcacc cccattgatc gagccctaac ccctgcccat caggcaatcc aattaagctc
      541 gcattgtctg ccttgtttag tttggctcct gcccgtttcg gcgtccactt gcacaaacac
      601 aaacaagcat tatatataag gctcgtctct ccctcccaac cacactcact tttttgcccg
      661 tcttcccttg ctaacacaaa agtcaagaac acaaacaacc accccaaccc ccttacacac
      721 aagacatatc tacagcaatg ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
      781 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
      841 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
      901 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
      961 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     1021 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     1081 cggtcgcgga ggcaatggat gccattgctg cggccgatct tagccagacg agcgggttcg
     1141 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     1201 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     1261 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     1321 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     1381 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     1441 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     1501 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     1561 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     1621 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     1681 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     1741 gcactcgtcc gagggcaaag gaataatcat gtaattagtt atgtcacgct tacattcacg
     1801 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt
     1861 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt
     1921 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga
     1981 gaaggttttg ggacgctcga gcaggcttgg aggcgacgtg gcagtggctc gcgggaggat
     2041 cggcgggaaa ctccgggata tccgtcgaga gtttacacgt gaatgggcag cgcaatccgt
     2101 tgacgacgat acgactggca aagtagcgac gatacctgcc agacaggtga catgtgcagg
     2161 ccgcactaac aaggaaacgg gcgctggggg gggcgggctt ctagactttg cccttgaaca
     2221 ggaatctagt gggggcttgt ctttccgcca atgggggagc gcctgttgag cgaccgtgca
     2281 tgctggaacg ccaagtgtat gtacagctgg tggtctcgca gcggtatgtg acgggactta
     2341 catctctcgt tttttcatga ccacgttttc acaggctcgg aggtacgtta aagttttgaa
     2401 ggctgcatct gaaccgaggt atgggggagt ttgaacagca acagtgttgg ggctgagggg
     2461 gccaagatcg gggcaagcag aggtcttaga tcaattgtgg ggatcccaaa gggctcgtta
     2521 tcaccttttt ccacccaatt cgggtcccaa ttgatccact actggcttgc ccaagttacc
     2581 ccagaaatgc cgccccggat ttctccaaaa acctaataag cttcatggaa cttggtggaa
     2641 gtgactttct acagagtgga gagaaccgtg gacacgtggc aatggcgctg accgtgtccc
     2701 cgagccgaat cgacgtgagg ggagaacgga gtatctgcgg tcatgtgacc ttccagagcg
     2761 gcgtcgccag tgtgcacgcg gtgaccccca gtttgggtct ctgtcacacg catactacct
     2821 cggctctcca catgctgaac tttatctttc gtggggatca taccgaaagt tgcaactacc
     2881 aggtgtatat aaagcctggt agactccccc cactttggac ctcatccaac caagacacac
     2941 aaaaatggac cgacttaaca acctcgccac ccagctcgag cagaaccccg ccaattcgta
     3001 gataatggaa tacaaatgga tatccagagt atacacatgg atagtataca ctgacacgac
     3061 aattctgtat ctctttatgt taactactgt gaggcattaa atagagcttg atatataaaa
     3121 tgttacattt cacagtctga acttttgcag attacctaat ttggtaagat attaattatg
     3181 aactgaaagt tgatggcatc cctaaatttg atgcaacgga atgcgtgcga tagtgaatca
     3241 ttgctaacag atcaagtaat cgtatcaatc gttccacgtc ttatccagac tcgatttccg
     3301 ccctaaccta aggaaactgc tgagtaattt cgaggttggt tcctaacaaa agccaagaaa
     3361 cgcgccgatg gtttagtggt aaaatccatc gttgccatcg atgggccccc ggttcgattc
     3421 cgggtcggcg caaagcaaga gagttagcgg aggttttaga gctagaaata gcaagttaaa
     3481 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtggtg ctttttttgt
     3541 gtgggtacgg tcttacctaa tcacacctgc cctcacatta ggcaccaacc taaacccaag
     3601 ctcaaccgtc gtcgactcag ccccgaagaa gcaggtactc gtgcaaatat ataacgaaca
     3661 gtttaacggc ggcccggaaa aagattcggt cgtcacgtga cctacctcca cccgactgaa
     3721 gagctcttca atgcagattt tcgtcaagac gctgaccggt aagaccatca cgcttgaggt
     3781 gagtacaacg atggctagtt taggggattt ccgccgccct gtgacgtaga cagggtggcg
     3841 tccaaacaca aacacacaca aacattacta acaacaggtc gagtcctccg acatcgcgtg
     3901 cattcgcggc cgcatttaaa tccccacttc agaagttcct atactttcta gagaatagga
     3961 acttcactat agagtcgaat aagggcgaca ccccataatt agcccgggcg aaaggcccag
     4021 tctttcgact gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcatgggg
     4081 agtccccaca ctaccatcgg cgctacggcg tttcacttct gagttcggca tggggtcagg
     4141 tgggaccacc gcgctactgc cgccaggcaa acaaggggtg ttatgagcca tattcaggta
     4201 taaatgggct cgcgataatg ttcagaattg gttaattggt tgtaacactg acccctattt
     4261 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4321 tgcttcaata atattgaaaa aggaagaata tgagtattca acatttccgt gtcgccctta
     4381 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4441 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4501 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4561 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4621 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4681 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4741 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4801 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     4861 taccaaacga cgagcgtgac accacgatgc ctgtagcgat ggcaacaacg ttgcgcaaac
     4921 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     4981 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5041 ataaatccgg agccggtgag cgtggttctc gcggtatcat cgcagcgctg gggccagatg
     5101 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5161 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa gcagagcatt
     5221 acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg
     5281 cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5341 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5401 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5461 gcagatacca aatactgttc ttctagtgta gccgtagtta gcccaccact tcaagaactc
     5521 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5581 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5641 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5701 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5761 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5821 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5881 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5941 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6001 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6061 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa ggcgagagta gggaactgcc
     6121 aggcatcaaa ctaagcagaa ggcccctgac ggatggcctt tttgcgtttc tacaaactct
     6181 ttctgtgttg taaaacgacg gccagtctta agctcgggcc ccctgggcgg ttctgataac
     6241 gagtaatcgt taatccgcaa ataacgtaaa aacccgcttc ggcgggtttt tttatggggg
     6301 gagtttaggg aaagagcatt tgtcagaata tttaagggcg cctgtcactt tgcttgatat
     6361 atgagaatta tttaacctta taaatgagaa aaaagcaacg cactttaaat aagatacgtt
     6421 gctttttcga ttgatgaaca cctataatta aactattcat ctattattta tgattttttg
     6481 tatatacaat atttctagtt tgttaaagag aattaagaaa ataaatctcg aaaataataa
     6541 agggaaaatc agtttttgat atcaaaatta tacatgtcaa cgataataca aaatataata
     6601 caaactataa gatgttatca gtatttatta tgcatttaga ataaattttg tgtcgccctt
     6661 attcgactca ctatagaagt tcctattctc tagaaagtat aggaacttca cttcatttta
     6721 tttaaatttg cggccgctat gtctgataaa aggatgtaac ataggcaagc tgctcgtgag
     6781 tgttgagtac gaaccttaga tccaaatcac ccgcacccac ggatatactt gcttgaatat
     6841 acagtagtaa ggagtttggc gcccgttttt tcgagcccca cacgtttcgg tgagtatgag
     6901 cggcggcaga ttcgagcgtt tccggtttcc gcggctggac gagagcccat gatgggggct
     6961 cccaccacca gcaatcaggg ccctgattac acacccacct gtaatgtcat gctgttcatc
     7021 gtggttaatg ctgctgtgtg ctgtgtgtgt gtgttgtttg gcgctcattg ttgcgttatg
     7081 cagcgtacac cacaatattg gaagcttatt agc
//
