LOCUS       sequence-428712-        6774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting to the gene 4HPPD locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6774)
  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 6774)
  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: pCfB12720
FEATURES             Location/Qualifiers
     source          1..6774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1963..1996)
                     /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(2466..2483)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2466..2482)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2581..2598)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2752..3340)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2832..2851)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3462..4253,4254..4322))
                     /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     4085..4104
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4323..4414)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4456..4475
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4481..4500
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4707..4740
                     /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(4804..4878)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     6530..6548
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(join(6565..6774,1..819))
                     /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(6565..6774,1..819))
                     /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 gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg
       61 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa
      121 gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc
      181 tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg
      241 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga
      301 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc
      361 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa
      421 cccgctcgtc tggctaagat cggccgcagc aatggcatcc attgcctccg cgaccggctg
      481 cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
      541 ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg
      601 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca
      661 gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc
      721 ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt
      781 ctcgacagac gtcgcggtga gttcaggctt tttacccatt gctgtagata tgtcttgtgt
      841 gtaagggggt tggggtggtt gtttgtgttc ttgacttttg tgttagcaag ggaagacggg
      901 caaaaaagtg agtgtggttg ggagggagag acgagcctta tatataatgc ttgtttgtgt
      961 ttgtgcaagt ggacgccgaa acgggcagga gccaaactaa acaaggcaga caatgcgagc
     1021 ttaattggat tgcctgatgg gcaggggtta gggctcgatc aatgggggtg cgaagtgaca
     1081 aaattgggaa ttaggttcgc aagcaaggct gacaagactt tggcccaaac atttgtacgc
     1141 ggtggacaac aggagccacc catcgtctgt cacgggctag ccggtcgtgc gtcctgtcag
     1201 gctccaccta ggctccatgc cactccatac aatcccacta gtgtaccgct aggccgcttt
     1261 tagctcccat ctaagacccc cccaaaacct ccactgtaca gtgcactgta ctgtgtggcg
     1321 atcaagggca agggaaaaaa ggcgcaaaca tgcatgcatg gaatgacgta ggtaaggcgt
     1381 tactagactg aaaagtggca catttcggcg tgccaaaggg tcctaggtgc gtttcgcgag
     1441 ctgggcgcca ggccaagccg ctccaaaacg cctctccgac tccctccagc ggcctccata
     1501 tccccatccc tctccacagc aatgttgtta agccttgcaa acgaaaaaat agaaaggcta
     1561 ataagcttcc aatattgtgg tgtacgctgc ataacgcaac aatgagcgcc aaacaacaca
     1621 cacacacagc acacagcagc attaaccacg atgaacagca tgacattaca ggtgggtgtg
     1681 taatcagggc cctgattgct ggtggtggga gcccccatca tgggctctcg tccagccgcg
     1741 gaaaccggaa acgctcgaat ctgccgccgc tcatactcac cgaaacgtgt ggggctcgaa
     1801 aaaacgggcg ccaaactcct tatactactg tatattcaag caagtatatc cgtgggtgcg
     1861 ggtgatttgg atctaaggtt cgtactcaac actcacgagc agcttgccta tgttacatcc
     1921 ttttatcaga catagcggcc gcaaatttaa ataaaatgaa gtgaagttcc tatactttct
     1981 agagaatagg aacttctata gtgagtcgaa taagggcgac acaaaattta ttctaaatgc
     2041 ataataaata ctgataacat cttatagttt gtattatatt ttgtattatc gttgacatgt
     2101 ataattttga tatcaaaaac tgattttccc tttattattt tcgagattta ttttcttaat
     2161 tctctttaac aaactagaaa tattgtatat acaaaaaatc ataaataata gatgaatagt
     2221 ttaattatag gtgttcatca atcgaaaaag caacgtatct tatttaaagt gcgttgcttt
     2281 tttctcattt ataaggttaa ataattctca tatatcaagc aaagtgacag gcgcccttaa
     2341 atattctgac aaatgctctt tccctaaact ccccccataa aaaaacccgc cgaagcgggt
     2401 ttttacgtta tttgcggatt aacgattact cgttatcaga accgcccagg gggcccgagc
     2461 ttaagactgg ccgtcgtttt acaacacaga aagagtttgt agaaacgcaa aaaggccatc
     2521 cgtcaggggc cttctgctta gtttgatgcc tggcagttcc ctactctcgc cttccgcttc
     2581 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2641 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2701 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2761 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2821 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2881 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2941 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3001 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3061 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3121 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggg ctaactacgg
     3181 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3241 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3301 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3361 tacggggtct gacgctcagt ggaacgacgc gcgcgtaact cacgttaagg gattttggtc
     3421 atgagcttgc gccgtcccgt caagtcagcg taatgctctg cttaccaatg cttaatcagt
     3481 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3541 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc gatgataccg
     3601 cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc cggaagggcc
     3661 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3721 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc catcgctaca
     3781 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3841 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3901 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3961 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4021 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4081 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4141 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4201 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4261 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4321 atattcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4381 tacatatttg aatgtattta gaaaaataaa caaatagggg tcagtgttac aaccaattaa
     4441 ccaattctga acattatcgc gagcccattt atacctgaat atggctcata acaccccttg
     4501 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
     4561 acgccgtagc gccgatggta gtgtggggac tccccatgcg agagtaggga actgccaggc
     4621 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgcccgggc taattatggg
     4681 gtgtcgccct tattcgactc tatagtgaag ttcctattct ctagaaagta taggaacttc
     4741 tgaagtgggg atttaaatgc ggccgcgaat gcacgcgata ccgtacccac acaaaaaaag
     4801 caccaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc
     4861 tatttctagc tctaaaaccg tagtacttgc atgcgttatg cgccgacccg gaatcgaacc
     4921 gggggcccat cgatggcaac gatggatttt accactaaac catcggcgcg tttcttggct
     4981 tttgttagga accaacctcg aaattactca gcagtttcct taggttaggg cggaaatcga
     5041 gtctggataa gacgtggaac gattgatacg attacttgat ctgttagcaa tgattcacta
     5101 tcgcacgcat tccgttgcat caaatttagg gatgccatca actttcagtt cataattaat
     5161 atcttaccaa attaggtaat ctgcaaaagt tcagactgtg aaatgtaaca ttttatatat
     5221 caagctctat ttaatgcctc acagtagtta acataaagag atacagaatt gtcgtgtcag
     5281 tgtatactat ccatgtgtat actctggata tccatttgta ttccattatc tacgaattgg
     5341 cggggttctg ctcgagctgg gtggcgaggt tgttaagtcg gtccattttt gtgtgtcttg
     5401 gttggatgag gtccaaagtg gggggagtct accaggcttt atatacacct ggtagttgca
     5461 actttcggta tgatccccac gaaagataaa gttcagcatg tggagagccg aggtagtatg
     5521 cgtgtgacag agacccaaac tgggggtcac cgcgtgcaca ctggcgacgc cgctctggaa
     5581 ggtcacatga ccgcagatac tccgttctcc cctcacgtcg attcggctcg gggacacggt
     5641 cagcgccatt gccacgtgtc cacggttctc tccactctgt agaaagtcac ttccaccaag
     5701 ttccatgaag cttattaggt ttttggagaa atccggggcg gcatttctgg ggtaacttgg
     5761 gcaagccagt agtggatcaa ttgggacccg aattgggtgg aaaaaggtga taacgagccc
     5821 tttgggatcc ccacaattga tctaagacct ctgcttgccc cgatcttggc cccctcagcc
     5881 ccaacactgt tgctcttcaa actcccccat acctcggttc agatgcagcc ttcaaaactt
     5941 taacgtacct ccgagcctgt gaaaacgtgg tcatgaaaaa acgagagatg taagtcccgt
     6001 cacataccgc tgcgagacca ccagctgtac atacacttgg cgttccagca tgcacggtcg
     6061 ctcaacaggc gctcccccat tggcggaaag acaagccccc actagattcc tgttcaaggg
     6121 caaagtctag aagcccgccc cccccagcgc ccgtttcctt gttagtgcgg cctgcacatg
     6181 tcacctgtct ggcaggtatc gtcgctactt tgccagtcgt atcgtcgtca acggattgcg
     6241 ctgcccattc acgtgtaaac tctcgacgga tatcccggag tttcccgccg atcctcccgc
     6301 gagccactgc cacgtcgcct ccaagcctgc tcgagcgtcc caaaaccttc tcaagcaagg
     6361 ttttcagtat aatgttacat gcgtacacgc gtctgtacag aaaaaaaaga aaaatttgaa
     6421 atataaataa cgttcttaat actaacataa ctataaaaaa ataaataggg acctagactt
     6481 caggttgtct aactccttcc ttttcggtta gagcggatgt ggggggaggg cgtgaatgta
     6541 agcgtgacat aactaattac atgattattc ctttgccctc ggacgagtgc tggggcgtcg
     6601 gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg
     6661 ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc
     6721 ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttg
//
