LOCUS       sequence-428711-        6949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting to the intergradtion site E2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6949)
  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 6949)
  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: pCfB11787
FEATURES             Location/Qualifiers
     source          1..6949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(2051..2084)
                     /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(2554..2571)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2554..2570)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2669..2686)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2840..3428)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2920..2939)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3550..4341,4342..4410))
                     /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     4173..4192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4411..4502)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4544..4563
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4569..4588
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4795..4828
                     /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(4892..4966)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(join(6830..6949,1..909))
                     /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(6830..6949,1..909))
                     /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 cccggctccg gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa
       61 attgccgtca accaagctct gatagagttg gtcaagacca atgcggagca tatacgcccg
      121 gagccgcggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg tctgctgctc
      181 catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt gggaatcccc
      241 gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg tcaggacatt
      301 gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct cggcccaaag
      361 catcagctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg
      421 ccagtgatac acatggggat cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg
      481 accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat cggccgcagc
      541 aatggcatcc attgcctccg cgaccggctg cagaacagcg ggcagttcgg tttcaggcag
      601 gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa
      661 ttccccaatg tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac
      721 ataacgatct ttgtagaaac catcggcgca gctatttacc cgcaggacat atccacgccc
      781 tcctacatcg aagctgaaag cacgagattc ttcgccctcc gagagctgca tcaggtcgga
      841 gacgctgtcg aacttttcga tcagaaactt ctcgacagac gtcgcggtga gttcaggctt
      901 tttacccatt gctgtagata tgtcttgtgt gtaagggggt tggggtggtt gtttgtgttc
      961 ttgacttttg tgttagcaag ggaagacggg caaaaaagtg agtgtggttg ggagggagag
     1021 acgagcctta tatataatgc ttgtttgtgt ttgtgcaagt ggacgccgaa acgggcagga
     1081 gccaaactaa acaaggcaga caatgcgagc ttaattggat tgcctgatgg gcaggggtta
     1141 gggctcgatc aatgggggtg cgaagtgaca aaattgggaa ttaggttcgc aagcaaggct
     1201 gacaagactt tggcccaaac atttgtacgc ggtggacaac aggagccacc catcgtctgt
     1261 cacgggctag ccggtcgtgc gtcctgtcag gctccaccta ggctccatgc cactccatac
     1321 aatcccacta gtgtaccgct aggccgcttt tagctcccat ctaagacccc cccaaaacct
     1381 ccactgtaca gtgcactgta ctgtgtggcg atcaagggca agggaaaaaa ggcgcaaaca
     1441 tgcatgcatg gaatgacgta ggtaaggcgt tactagactg aaaagtggca catttcggcg
     1501 tgccaaaggg tcctaggtgc gtttcgcgag ctgggcgcca ggccaagccg ctccaaaacg
     1561 cctctccgac tccctccagc ggcctccata tccccatccc tctccacagc aatgttgtta
     1621 agccttgcaa acgaaaaaat agaaaggcta ataagcttcc aatattgtgg tgtacgctgc
     1681 ataacgcaac aatgagcgcc aaacaacaca cacacagcac acagcagcat taaccacgat
     1741 gaacagcatg acattacagg tgggtgtgta atcagggccc tgattgctgg tggtgggagc
     1801 ccccatcatg ggctctcgtc cagccgcgga aaccggaaac gctcgaatct gccgccgctc
     1861 atactcaccg aaacgtgtgg ggctcgaaaa aacgggcgcc aaactcctta tactactgta
     1921 tattcaagca agtatatccg tgggtgcggg tgatttggat ctaaggttcg tactcaacac
     1981 tcacgagcag cttgcctatg ttacatcctt ttatcagaca tagcggccgc aaatttaaat
     2041 aaaatgaagt gaagttccta tactttctag agaataggaa cttctatagt gagtcgaata
     2101 agggcgacac aaaatttatt ctaaatgcat aataaatact gataacatct tatagtttgt
     2161 attatatttt gtattatcgt tgacatgtat aattttgata tcaaaaactg attttccctt
     2221 tattattttc gagatttatt ttcttaattc tctttaacaa actagaaata ttgtatatac
     2281 aaaaaatcat aaataataga tgaatagttt aattataggt gttcatcaat cgaaaaagca
     2341 acgtatctta tttaaagtgc gttgcttttt tctcatttat aaggttaaat aattctcata
     2401 tatcaagcaa agtgacaggc gcccttaaat attctgacaa atgctctttc cctaaactcc
     2461 ccccataaaa aaacccgccg aagcgggttt ttacgttatt tgcggattaa cgattactcg
     2521 ttatcagaac cgcccagggg gcccgagctt aagactggcc gtcgttttac aacacagaaa
     2581 gagtttgtag aaacgcaaaa aggccatccg tcaggggcct tctgcttagt ttgatgcctg
     2641 gcagttccct actctcgcct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2701 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2761 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2821 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2881 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2941 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3001 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3061 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3121 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3181 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3241 agttcttgaa gtggtgggct aactacggct acactagaag aacagtattt ggtatctgcg
     3301 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3361 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3421 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgacgcgc
     3481 gcgtaactca cgttaaggga ttttggtcat gagcttgcgc cgtcccgtca agtcagcgta
     3541 atgctctgct taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3601 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3661 atctggcccc agcgctgcga tgataccgcg agaaccacgc tcaccggctc cggatttatc
     3721 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3781 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3841 tttgcgcaac gttgttgcca tcgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3901 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3961 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4021 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4081 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4141 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4201 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4261 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4321 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4381 aagggcgaca cggaaatgtt gaatactcat attcttcctt tttcaatatt attgaagcat
     4441 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4501 aataggggtc agtgttacaa ccaattaacc aattctgaac attatcgcga gcccatttat
     4561 acctgaatat ggctcataac accccttgtt tgcctggcgg cagtagcgcg gtggtcccac
     4621 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggactc
     4681 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
     4741 tgggcctttc gcccgggcta attatggggt gtcgccctta ttcgactcta tagtgaagtt
     4801 cctattctct agaaagtata ggaacttctg aagtggggat ttaaatgcgg ccgcgaatgc
     4861 acgcgatacc gtacccacac aaaaaaagca ccaccgactc ggtgccactt tttcaagttg
     4921 ataacggact agccttattt taacttgcta tttctagctc taaaaccact cgccagcact
     4981 tattggtgcg ccgacccgga atcgaaccgg gggcccatcg atggcaacga tggattttac
     5041 cactaaacca tcggcgcgtt tcttggcttt tgttaggaac caacctcgaa attactcagc
     5101 agtttcctta ggttagggcg gaaatcgagt ctggataaga cgtggaacga ttgatacgat
     5161 tacttgatct gttagcaatg attcactatc gcacgcattc cgttgcatca aatttaggga
     5221 tgccatcaac tttcagttca taattaatat cttaccaaat taggtaatct gcaaaagttc
     5281 agactgtgaa atgtaacatt ttatatatca agctctattt aatgcctcac agtagttaac
     5341 ataaagagat acagaattgt cgtgtcagtg tatactatcc atgtgtatac tctggatatc
     5401 catttgtatt ccattatcta cgaattggcg gggttctgct cgagctgggt ggcgaggttg
     5461 ttaagtcggt ccatttttgt gtgtcttggt tggatgaggt ccaaagtggg gggagtctac
     5521 caggctttat atacacctgg tagttgcaac tttcggtatg atccccacga aagataaagt
     5581 tcagcatgtg gagagccgag gtagtatgcg tgtgacagag acccaaactg ggggtcaccg
     5641 cgtgcacact ggcgacgccg ctctggaagg tcacatgacc gcagatactc cgttctcccc
     5701 tcacgtcgat tcggctcggg gacacggtca gcgccattgc cacgtgtcca cggttctctc
     5761 cactctgtag aaagtcactt ccaccaagtt ccatgaagct tattaggttt ttggagaaat
     5821 ccggggcggc atttctgggg taacttgggc aagccagtag tggatcaatt gggacccgaa
     5881 ttgggtggaa aaaggtgata acgagccctt tgggatcccc acaattgatc taagacctct
     5941 gcttgccccg atcttggccc cctcagcccc aacactgttg ctcttcaaac tcccccatac
     6001 ctcggttcag atgcagcctt caaaacttta acgtacctcc gagcctgtga aaacgtggtc
     6061 atgaaaaaac gagagatgta agtcccgtca cataccgctg cgagaccacc agctgtacat
     6121 acacttggcg ttccagcatg cacggtcgct caacaggcgc tcccccattg gcggaaagac
     6181 aagcccccac tagattcctg ttcaagggca aagtctagaa gcccgccccc cccagcgccc
     6241 gtttccttgt tagtgcggcc tgcacatgtc acctgtctgg caggtatcgt cgctactttg
     6301 ccagtcgtat cgtcgtcaac ggattgcgct gcccattcac gtgtaaactc tcgacggata
     6361 tcccggagtt tcccgccgat cctcccgcga gccactgcca cgtcgcctcc aagcctgctg
     6421 aattcggaca cgggcatctc acttgcgtat gtatggaaaa taaaaatacg acagtcgaac
     6481 ataaagggat gacgagtagt cacagaggga attgatgaac aaagacggga ttttgccacc
     6541 tacaagccag attttctatt tacaacaata tctggtcaaa tttcagtttc gttacataaa
     6601 tcgttatgtc aaaggagtgt gggaggttaa gagaattatc accggcaaac tatctgttaa
     6661 ttgcctaaat gccaacaccg ttgtaggcaa cagcgttggg agagcccttg aggttggtgg
     6721 tgggaagaac accggtgttg gactcagtaa taagagcttc gacctgggca ggggtgagaa
     6781 cctcgtcatt gatggacagg tagtaggagg caagaccggc aacgtggggt tattcctttg
     6841 ccctcggacg agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat
     6901 cggtccagac ggccgcgctt ctgcgggcga tttgtgtacg cccgacagt
//
