LOCUS       sequence-428713-        6783 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting to the intergradtion site F3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6783)
  AUTHORS   Jiang W, Wang S, Avila P, Jorgensen TS, Yang Z, Borodina I
  TITLE     Combinatorial iterative method for metabolic engineering of Yarrowia
            lipolytica: application for betanin biosynthesis.
  JOURNAL   Metab Eng. 2024 Sep 9:S1096-7176(24)00110-1. doi: 
            10.1016/j.ymben.2024.09.003.
  PUBMED    39260817
REFERENCE   2  (bases 1 to 6783)
  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: "Metab Eng. 
            2024 Sep 9:S1096-7176(24)00110-1. doi: 10.1016/j.ymben.2024.09.003."
COMMENT     Sequence Label: pCfB12719
FEATURES             Location/Qualifiers
     source          1..6783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1968..2001)
                     /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(2471..2488)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2471..2487)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2586..2603)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2757..3345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2837..2856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3467..4258,4259..4327))
                     /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     4090..4109
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4328..4419)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4461..4480
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4486..4505
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4712..4745
                     /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)"
     misc_RNA        complement(4809..4883)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     6544..6562
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(join(6579..6783,1..824))
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(join(6579..6783,1..824))
                     /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"
ORIGIN
        1 agttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc tgcaagctcc
       61 ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag
      121 aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg
      181 accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg
      241 aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg
      301 acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca
      361 atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg
      421 ccgaacccgc tcgtctggct aagatcggcc gcagcaatgg catccattgc ctccgcgacc
      481 ggctgcagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac accctgtgca
      541 cggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag cacttccgga
      601 atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg
      661 gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga
      721 gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga
      781 aacttctcga cagacgtcgc ggtgagttca ggctttttac ccattgctgt agatatgtct
      841 tgtgtgtaag ggggttgggg tggttgtttg tgttcttgac ttttgtgtta gcaagggaag
      901 acgggcaaaa aagtgagtgt ggttgggagg gagagacgag ccttatatat aatgcttgtt
      961 tgtgtttgtg caagtggacg ccgaaacggg caggagccaa actaaacaag gcagacaatg
     1021 cgagcttaat tggattgcct gatgggcagg ggttagggct cgatcaatgg gggtgcgaag
     1081 tgacaaaatt gggaattagg ttcgcaagca aggctgacaa gactttggcc caaacatttg
     1141 tacgcggtgg acaacaggag ccacccatcg tctgtcacgg gctagccggt cgtgcgtcct
     1201 gtcaggctcc acctaggctc catgccactc catacaatcc cactagtgta ccgctaggcc
     1261 gcttttagct cccatctaag acccccccaa aacctccact gtacagtgca ctgtactgtg
     1321 tggcgatcaa gggcaaggga aaaaaggcgc aaacatgcat gcatggaatg acgtaggtaa
     1381 ggcgttacta gactgaaaag tggcacattt cggcgtgcca aagggtccta ggtgcgtttc
     1441 gcgagctggg cgccaggcca agccgctcca aaacgcctct ccgactccct ccagcggcct
     1501 ccatatcccc atccctctcc acagcaatgt tgttaagcct tgcaaacgaa aaaatagaaa
     1561 ggctaataag cttccaatat tgtggtgtac gctgcataac gcaacaatga gcgccaaaca
     1621 acacacacac acagcacaca gcagcattaa ccacgatgaa cagcatgaca ttacaggtgg
     1681 gtgtgtaatc agggccctga ttgctggtgg tgggagcccc catcatgggc tctcgtccag
     1741 ccgcggaaac cggaaacgct cgaatctgcc gccgctcata ctcaccgaaa cgtgtggggc
     1801 tcgaaaaaac gggcgccaaa ctccttatac tactgtatat tcaagcaagt atatccgtgg
     1861 gtgcgggtga tttggatcta aggttcgtac tcaacactca cgagcagctt gcctatgtta
     1921 catcctttta tcagacatag cggccgcaaa tttaaataaa atgaagtgaa gttcctatac
     1981 tttctagaga ataggaactt ctatagtgag tcgaataagg gcgacacaaa atttattcta
     2041 aatgcataat aaatactgat aacatcttat agtttgtatt atattttgta ttatcgttga
     2101 catgtataat tttgatatca aaaactgatt ttccctttat tattttcgag atttattttc
     2161 ttaattctct ttaacaaact agaaatattg tatatacaaa aaatcataaa taatagatga
     2221 atagtttaat tataggtgtt catcaatcga aaaagcaacg tatcttattt aaagtgcgtt
     2281 gcttttttct catttataag gttaaataat tctcatatat caagcaaagt gacaggcgcc
     2341 cttaaatatt ctgacaaatg ctctttccct aaactccccc cataaaaaaa cccgccgaag
     2401 cgggttttta cgttatttgc ggattaacga ttactcgtta tcagaaccgc ccagggggcc
     2461 cgagcttaag actggccgtc gttttacaac acagaaagag tttgtagaaa cgcaaaaagg
     2521 ccatccgtca ggggccttct gcttagtttg atgcctggca gttccctact ctcgccttcc
     2581 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2641 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2701 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2761 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2821 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2881 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2941 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3001 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3061 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3121 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtgggctaac
     3181 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3241 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3301 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3361 ttttctacgg ggtctgacgc tcagtggaac gacgcgcgcg taactcacgt taagggattt
     3421 tggtcatgag cttgcgccgt cccgtcaagt cagcgtaatg ctctgcttac caatgcttaa
     3481 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3541 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagc gctgcgatga
     3601 taccgcgaga accacgctca ccggctccgg atttatcagc aataaaccag ccagccggaa
     3661 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3721 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccatcg
     3781 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3841 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3901 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3961 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4021 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4081 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4141 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4201 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4261 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4321 tactcatatt cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4381 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggtcagt gttacaacca
     4441 attaaccaat tctgaacatt atcgcgagcc catttatacc tgaatatggc tcataacacc
     4501 ccttgtttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa
     4561 gtgaaacgcc gtagcgccga tggtagtgtg gggactcccc atgcgagagt agggaactgc
     4621 caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgcc cgggctaatt
     4681 atggggtgtc gcccttattc gactctatag tgaagttcct attctctaga aagtatagga
     4741 acttctgaag tggggattta aatgcggccg cgaatgcacg cgataccgta cccacacaaa
     4801 aaaagcacca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa
     4861 cttgctattt ctagctctaa aacccgtgaa attatcggag aaataaccaa cctgcgccga
     4921 cccggaatcg aaccgggggc ccatcgatgg caacgatgga ttttaccact aaaccatcgg
     4981 cgcgtttctt ggcttttgtt aggaaccaac ctcgaaatta ctcagcagtt tccttaggtt
     5041 agggcggaaa tcgagtctgg ataagacgtg gaacgattga tacgattact tgatctgtta
     5101 gcaatgattc actatcgcac gcattccgtt gcatcaaatt tagggatgcc atcaactttc
     5161 agttcataat taatatctta ccaaattagg taatctgcaa aagttcagac tgtgaaatgt
     5221 aacattttat atatcaagct ctatttaatg cctcacagta gttaacataa agagatacag
     5281 aattgtcgtg tcagtgtata ctatccatgt gtatactctg gatatccatt tgtattccat
     5341 tatctacgaa ttggcggggt tctgctcgag ctgggtggcg aggttgttaa gtcggtccat
     5401 ttttgtgtgt cttggttgga tgaggtccaa agtgggggga gtctaccagg ctttatatac
     5461 acctggtagt tgcaactttc ggtatgatcc ccacgaaaga taaagttcag catgtggaga
     5521 gccgaggtag tatgcgtgtg acagagaccc aaactggggg tcaccgcgtg cacactggcg
     5581 acgccgctct ggaaggtcac atgaccgcag atactccgtt ctcccctcac gtcgattcgg
     5641 ctcggggaca cggtcagcgc cattgccacg tgtccacggt tctctccact ctgtagaaag
     5701 tcacttccac caagttccat gaagcttatt aggtttttgg agaaatccgg ggcggcattt
     5761 ctggggtaac ttgggcaagc cagtagtgga tcaattggga cccgaattgg gtggaaaaag
     5821 gtgataacga gccctttggg atccccacaa ttgatctaag acctctgctt gccccgatct
     5881 tggccccctc agccccaaca ctgttgctct tcaaactccc ccatacctcg gttcagatgc
     5941 agccttcaaa actttaacgt acctccgagc ctgtgaaaac gtggtcatga aaaaacgaga
     6001 gatgtaagtc ccgtcacata ccgctgcgag accaccagct gtacatacac ttggcgttcc
     6061 agcatgcacg gtcgctcaac aggcgctccc ccattggcgg aaagacaagc ccccactaga
     6121 ttcctgttca agggcaaagt ctagaagccc gcccccccca gcgcccgttt ccttgttagt
     6181 gcggcctgca catgtcacct gtctggcagg tatcgtcgct actttgccag tcgtatcgtc
     6241 gtcaacggat tgcgctgccc attcacgtgt aaactctcga cggatatccc ggagtttccc
     6301 gccgatcctc ccgcgagcca ctgccacgtc gcctccaagc ctgctcgagc gtcccaaaac
     6361 cttctcaagc aaggttttca gtataatgtt acatgcgtac acgcgtctgt acagaaaaaa
     6421 aagaaaaatt tgaaatataa ataacgttct taatactaac ataactataa aaaaataaat
     6481 agggacctag acttcaggtt gtctaactcc ttccttttcg gttagagcgg atgtgggggg
     6541 agggcgtgaa tgtaagcgtg acataactaa ttacatgatt attcctttgc cctcggacga
     6601 gtgctggggc gtcggtttcc actatcggcg agtacttcta cacagccatc ggtccagacg
     6661 gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt
     6721 gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac caagctctga
     6781 tag
//
