LOCUS       sequence-435385-        7176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The base plasmid of TUNEYALI for TF22.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7176)
  TITLE     Regulating transcription factors in Yarrowia lipolytica
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7176)
  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            "Unpublished"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: pCfb13212
FEATURES             Location/Qualifiers
     source          1..7176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             472..1500
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             472..1500
                     /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(1517..1535)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     misc_RNA        3187..3261
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3729..3762)
                     /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(3969..3988)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3994..4013)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4055..4146
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4147..4215,4216..5007)
                     /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(4365..4384)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5129..5717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5618..5637
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5871..5888
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5986..6003
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5987..6003
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6473..6506
                     /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 aggttttggg ggggtcttag atgggagcta aaagcggcct agcggtacac tagtgggatt
       61 gtatggagtg gcatggagcc taggtggagc ctgacaggac gcacgaccgg ctagcccgtg
      121 acagacgatg ggtggctcct gttgtccacc gcgtacaaat gtttgggcca aagtcttgtc
      181 agccttgctt gcgaacctaa ttcccaattt tgtcacttcg cacccccatt gatcgagccc
      241 taacccctgc ccatcaggca atccaattaa gctcgcattg tctgccttgt ttagtttggc
      301 tcctgcccgt ttcggcgtcc acttgcacaa acacaaacaa gcattatata taaggctcgt
      361 ctctccctcc caaccacact cacttttttg cccgtcttcc cttgctaaca caaaagtcaa
      421 gaacacaaac aaccacccca acccccttac acacaagaca tatctacagc aatgggtaaa
      481 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
      541 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
      601 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
      661 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
      721 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
      781 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggcaat ggatgccatt
      841 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
      901 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
      961 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     1021 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     1081 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     1141 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     1201 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     1261 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     1321 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     1381 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     1441 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaataa
     1501 tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg
     1561 aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt
     1621 tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt
     1681 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgagcaggc
     1741 ttggaggcga cgtggcagtg gctcgcggga ggatcggcgg gaaactccgg gatatccgtc
     1801 gagagtttac acgtgaatgg gcagcgcaat ccgttgacga cgatacgact ggcaaagtag
     1861 cgacgatacc tgccagacag gtgacatgtg caggccgcac taacaaggaa acgggcgctg
     1921 gggggggcgg gcttctagac tttgcccttg aacaggaatc tagtgggggc ttgtctttcc
     1981 gccaatgggg gagcgcctgt tgagcgaccg tgcatgctgg aacgccaagt gtatgtacag
     2041 ctggtggtct cgcagcggta tgtgacggga cttacatctc tcgttttttc atgaccacgt
     2101 tttcacaggc tcggaggtac gttaaagttt tgaaggctgc atctgaaccg aggtatgggg
     2161 gagtttgaac agcaacagtg ttggggctga gggggccaag atcggggcaa gcagaggtct
     2221 tagatcaatt gtggggatcc caaagggctc gttatcacct ttttccaccc aattcgggtc
     2281 ccaattgatc cactactggc ttgcccaagt taccccagaa atgccgcccc ggatttctcc
     2341 aaaaacctaa taagcttcat ggaacttggt ggaagtgact ttctacagag tggagagaac
     2401 cgtggacacg tggcaatggc gctgaccgtg tccccgagcc gaatcgacgt gaggggagaa
     2461 cggagtatct gcggtcatgt gaccttccag agcggcgtcg ccagtgtgca cgcggtgacc
     2521 cccagtttgg gtctctgtca cacgcatact acctcggctc tccacatgct gaactttatc
     2581 tttcgtgggg atcataccga aagttgcaac taccaggtgt atataaagcc tggtagactc
     2641 cccccacttt ggacctcatc caaccaagac acacaaaaat ggaccgactt aacaacctcg
     2701 ccacccagct cgagcagaac cccgccaatt cgtagataat ggaatacaaa tggatatcca
     2761 gagtatacac atggatagta tacactgaca cgacaattct gtatctcttt atgttaacta
     2821 ctgtgaggca ttaaatagag cttgatatat aaaatgttac atttcacagt ctgaactttt
     2881 gcagattacc taatttggta agatattaat tatgaactga aagttgatgg catccctaaa
     2941 tttgatgcaa cggaatgcgt gcgatagtga atcattgcta acagatcaag taatcgtatc
     3001 aatcgttcca cgtcttatcc agactcgatt tccgccctaa cctaaggaaa ctgctgagta
     3061 atttcgaggt tggttcctaa caaaagccaa gaaacgcgcc gatggtttag tggtaaaatc
     3121 catcgttgcc atcgatgggc ccccggttcg attccgggtc ggcgcaatcc aaattagaga
     3181 ccaaaggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     3241 aaaagtggca ccgagtcggt ggtgcttttt ttgtgtgggt acggtcgagg ccaccccccg
     3301 aaactacctg caggagatgt ccgagaaccc caccaagcgg ttccaggccc tggaggactc
     3361 caagaaggag gagtttgtca agtaccgacg acgataaatg tatatagtaa catatttgat
     3421 attgagagtg tgaaatgtag atgtgtggac tgaagagctc ttcaatgaca cgatcaaggc
     3481 gtcctcggcc ctcctacgtg atggagggcg acgactttgc gggtgacgac tcggactcgg
     3541 acttcaaccc cggcattgaa ccgaccgtgg aagatgacga catggacgtg gatgtgaacg
     3601 aggtcaaggc tgaggaggaa gatggcatcg cgtgcattcg cggccgcatc gcgtgcattc
     3661 gcggccgcat cgcgtgcatt cgcggccgca tcgcgtgcat tcgcggccgc atttaaatcc
     3721 ccacttcaga agttcctata ctttctagag aataggaact tcactataga gtcgaataag
     3781 ggcgacaccc cataattagc ccgggcgaaa ggcccagtct ttcgactgag cctttcgttt
     3841 tatttgatgc ctggcagttc cctactctcg catggggagt ccccacacta ccatcggcgc
     3901 tacggcgttt cacttctgag ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc
     3961 caggcaaaca aggggtgtta tgagccatat tcaggtataa atgggctcgc gataatgttc
     4021 agaattggtt aattggttgt aacactgacc cctatttgtt tatttttcta aatacattca
     4081 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4141 aagaatatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     4201 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     4261 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     4321 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     4381 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     4441 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     4501 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4561 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4621 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4681 acgatgcctg tagcgatggc aacaacgttg cgcaaactat taactggcga actacttact
     4741 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4801 ctgcgctcgg cccttccggc tggctggttt attgctgata aatccggagc cggtgagcgt
     4861 ggttctcgcg gtatcatcgc agcgctgggg ccagatggta agccctcccg tatcgtagtt
     4921 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4981 ggtgcctcac tgattaagca ttggtaagca gagcattacg ctgacttgac gggacggcgc
     5041 aagctcatga ccaaaatccc ttaacgtgag ttacgcgcgc gtcgttccac tgagcgtcag
     5101 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5161 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5221 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5281 tagtgtagcc gtagttagcc caccacttca agaactctgt agcaccgcct acatacctcg
     5341 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5401 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5461 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5521 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5581 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5641 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5701 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5761 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5821 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5881 tgagcgagga agcggaaggc gagagtaggg aactgccagg catcaaacta agcagaaggc
     5941 ccctgacgga tggccttttt gcgtttctac aaactctttc tgtgttgtaa aacgacggcc
     6001 agtcttaagc tcgggccccc tgggcggttc tgataacgag taatcgttaa tccgcaaata
     6061 acgtaaaaac ccgcttcggc gggttttttt atggggggag tttagggaaa gagcatttgt
     6121 cagaatattt aagggcgcct gtcactttgc ttgatatatg agaattattt aaccttataa
     6181 atgagaaaaa agcaacgcac tttaaataag atacgttgct ttttcgattg atgaacacct
     6241 ataattaaac tattcatcta ttatttatga ttttttgtat atacaatatt tctagtttgt
     6301 taaagagaat taagaaaata aatctcgaaa ataataaagg gaaaatcagt ttttgatatc
     6361 aaaattatac atgtcaacga taatacaaaa tataatacaa actataagat gttatcagta
     6421 tttattatgc atttagaata aattttgtgt cgcccttatt cgactcacta tagaagttcc
     6481 tattctctag aaagtatagg aacttcactt cattttattt aaatttgcgg ccgctatgtc
     6541 tgataaaagg atgtaacata ggcaagctgc tcgtgagtgt tgagtacgaa ccttagatcc
     6601 aaatcacccg cacccacgga tatacttgct tgaatataca gtagtaagga gtttggcgcc
     6661 cgttttttcg agccccacac gtttcggtga gtatgagcgg cggcagattc gagcgtttcc
     6721 ggtttccgcg gctggacgag agcccatgat gggggctccc accaccagca atcagggccc
     6781 tgattacaca cccacctgta atgtcatgct gttcatcgtg gttaatgctg ctgtgtgctg
     6841 tgtgtgtgtg ttgtttggcg ctcattgttg cgttatgcag cgtacaccac aatattggaa
     6901 gcttattagc ctttctattt tttcgtttgc aaggcttaac aacattgctg tggagaggga
     6961 tggggatatg gaggccgctg gagggagtcg gagaggcgtt ttggagcggc ttggcctggc
     7021 gcccagctcg cgaaacgcac ctaggaccct ttggcacgcc gaaatgtgcc acttttcagt
     7081 ctagtaacgc cttacctacg tcattccatg catgcatgtt tgcgcctttt ttcccttgcc
     7141 cttgatcgcc acacagtaca gtgcactgta cagtgg
//
