LOCUS       sequence-424298-        6960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting to the intergradtion site E3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6960)
  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 6960)
  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: pCfB8860
FEATURES             Location/Qualifiers
     source          1..6960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        1254..1328
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1392..1425)
                     /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(1632..1651)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1657..1676)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1718..1809
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1810..1878,1879..2670)
                     /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(2028..2047)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2792..3380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3281..3300
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3534..3551
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3649..3666
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3650..3666
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4136..4169
                     /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)"
     CDS             5313..6341
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             5313..6341
                     /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 tgcccttgaa caggaatcta gtgggggctt gtctttccgc caatggggga gcgcctgttg
       61 agcgaccgtg catgctggaa cgccaagtgt atgtacagct ggtggtctcg cagcggtatg
      121 tgacgggact tacatctctc gttttttcat gaccacgttt tcacaggctc ggaggtacgt
      181 taaagttttg aaggctgcat ctgaaccgag gtatggggga gtttgaagag caacagtgtt
      241 ggggctgagg gggccaagat cggggcaagc agaggtctta gatcaattgt ggggatccca
      301 aagggctcgt tatcaccttt ttccacccaa ttcgggtccc aattgatcca ctactggctt
      361 gcccaagtta ccccagaaat gccgccccgg atttctccaa aaacctaata agcttcatgg
      421 aacttggtgg aagtgacttt ctacagagtg gagagaaccg tggacacgtg gcaatggcgc
      481 tgaccgtgtc cccgagccga atcgacgtga ggggagaacg gagtatctgc ggtcatgtga
      541 ccttccagag cggcgtcgcc agtgtgcacg cggtgacccc cagtttgggt ctctgtcaca
      601 cgcatactac ctcggctctc cacatgctga actttatctt tcgtggggat cataccgaaa
      661 gttgcaacta ccaggtgtat ataaagcctg gtagactccc cccactttgg acctcatcca
      721 accaagacac acaaaaatgg accgacttaa caacctcgcc acccagctcg agcagaaccc
      781 cgccaattcg tagataatgg aatacaaatg gatatccaga gtatacacat ggatagtata
      841 cactgacacg acaattctgt atctctttat gttaactact gtgaggcatt aaatagagct
      901 tgatatataa aatgttacat ttcacagtct gaacttttgc agattaccta atttggtaag
      961 atattaatta tgaactgaaa gttgatggca tccctaaatt tgatgcaacg gaatgcgtgc
     1021 gatagtgaat cattgctaac agatcaagta atcgtatcaa tcgttccacg tcttatccag
     1081 actcgatttc cgccctaacc taaggaaact gctgagtaat ttcgaggttg gttcctaaca
     1141 aaagccaaga aacgcgccga tggtttagtg gtaaaatcca tcgttgccat cgatgggccc
     1201 ccggttcgat tccgggtcgg cgcaggttgg ttaccttgaa tcagtcgatg gtcgttttag
     1261 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     1321 agtcggtggt gctttttttg tgtgggtacg gtatcgcgtg cattcgcggc cgcatttaaa
     1381 tccccacttc agaagttcct atactttcta gagaatagga acttcactat agagtcgaat
     1441 aagggcgaca ccccataatt agcccgggcg aaaggcccag tctttcgact gagcctttcg
     1501 ttttatttga tgcctggcag ttccctactc tcgcatgggg agtccccaca ctaccatcgg
     1561 cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc gcgctactgc
     1621 cgccaggcaa acaaggggtg ttatgagcca tattcaggta taaatgggct cgcgataatg
     1681 ttcagaattg gttaattggt tgtaacactg acccctattt gtttattttt ctaaatacat
     1741 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1801 aggaagaata tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1861 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1921 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1981 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2041 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2101 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2161 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2221 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2281 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2341 accacgatgc ctgtagcgat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2401 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2461 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatccgg agccggtgag
     2521 cgtggttctc gcggtatcat cgcagcgctg gggccagatg gtaagccctc ccgtatcgta
     2581 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2641 ataggtgcct cactgattaa gcattggtaa gcagagcatt acgctgactt gacgggacgg
     2701 cgcaagctca tgaccaaaat cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt
     2761 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2821 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2881 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     2941 ttctagtgta gccgtagtta gcccaccact tcaagaactc tgtagcaccg cctacatacc
     3001 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3061 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3121 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3181 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3241 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3301 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3361 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3421 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3481 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3541 cagtgagcga ggaagcggaa ggcgagagta gggaactgcc aggcatcaaa ctaagcagaa
     3601 ggcccctgac ggatggcctt tttgcgtttc tacaaactct ttctgtgttg taaaacgacg
     3661 gccagtctta agctcgggcc ccctgggcgg ttctgataac gagtaatcgt taatccgcaa
     3721 ataacgtaaa aacccgcttc ggcgggtttt tttatggggg gagtttaggg aaagagcatt
     3781 tgtcagaata tttaagggcg cctgtcactt tgcttgatat atgagaatta tttaacctta
     3841 taaatgagaa aaaagcaacg cactttaaat aagatacgtt gctttttcga ttgatgaaca
     3901 cctataatta aactattcat ctattattta tgattttttg tatatacaat atttctagtt
     3961 tgttaaagag aattaagaaa ataaatctcg aaaataataa agggaaaatc agtttttgat
     4021 atcaaaatta tacatgtcaa cgataataca aaatataata caaactataa gatgttatca
     4081 gtatttatta tgcatttaga ataaattttg tgtcgccctt attcgactca ctatagaagt
     4141 tcctattctc tagaaagtat aggaacttca cttcatttta tttaaatttg cggccgctat
     4201 gtctgataaa aggatgtaac ataggcaagc tgctcgtgag tgttgagtac gaaccttaga
     4261 tccaaatcac ccgcacccac ggatatactt gcttgaatat acagtagtat aaggagtttg
     4321 gcgcccgttt tttcgagccc cacacgtttc ggtgagtatg agcggcggca gattcgagcg
     4381 tttccggttt ccgcggctgg acgagagccc atgatggggg ctcccaccac cagcaatcag
     4441 ggccctgatt acacacccac ctgtaatgtc atgctgttca tcgtggttaa tgctgctgtg
     4501 tgctgtgtgt gtgtgttgtt tggcgctcat tgttgcgtta tgcagcgtac accacaatat
     4561 tggaagctta ttagcctttc tattttttcg tttgcaaggc ttaacaacat tgctgtggag
     4621 agggatgggg atatggaggc cgctggaggg agtcggagag gcgttttgga gcggcttggc
     4681 ctggcgccca gctcgcgaaa cgcacctagg accctttggc acgccgaaat gtgccacttt
     4741 tcagtctagt aacgccttac ctacgtcatt ccatgcatgc atgtttgcgc cttttttccc
     4801 ttgcccttga tcgccacaca gtacagtgca ctgtacagtg gaggttttgg gggggtctta
     4861 gatgggagct aaaagcggcc tagcggtaca ctagtgggat tgtatggagt ggcatggagc
     4921 ctaggtggag cctgacagga cgcacgaccg gctagcccgt gacagacgat gggtggctcc
     4981 tgttgtccac cgcgtacaaa tgtttgggcc aaagtcttgt cagccttgct tgcgaaccta
     5041 attcccaatt ttgtcacttc gcacccccat tgatcgagcc ctaacccctg cccatcaggc
     5101 aatccaatta agctcgcatt gtctgccttg tttagtttgg ctcctgcccg tttcggcgtc
     5161 cacttgcaca aacacaaaca agcattatat ataaggctcg tctctccctc ccaaccacac
     5221 tcactttttt gcccgtcttc ccttgctaac acaaaagtca agaacacaaa caaccacccc
     5281 aaccccctta cacacaagac atatctacag caatgggtaa aaagcctgaa ctcaccgcga
     5341 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
     5401 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     5461 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     5521 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct
     5581 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
     5641 ccgctgttct gcagccggtc gcggaggcaa tggatgccat tgctgcggcc gatcttagcc
     5701 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     5761 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     5821 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     5881 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     5941 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     6001 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     6061 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     6121 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     6181 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     6241 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     6301 gaaaccgacg ccccagcact cgtccgaggg caaaggaata accccacgtt gccggtcttg
     6361 cctcctacta cctgtccatc aatgacgagg ttctcacccc tgcccaggtc gaagctctta
     6421 ttactgagtc caacaccggt gttcttccca ccaccaacct caagggctct cccaacgctg
     6481 ttgcctacaa cggtgttggc atttaggcaa ttaacagata gtttgccggt gataattctc
     6541 ttaacctccc acactccttt gacataacga tttatgtaac gaaactgaaa tttgaccaga
     6601 tattgttgta aatagaaaat ctggcttgta ggtggcaaaa tcccgtcttt gttcatcaat
     6661 tccctctgtg actactcgtc atccctttat gttcgactgt cgtattttta ttttccatac
     6721 atacgcaagt gagatgcccg tgtccgaatt cagcaggctt ggaggcgacg tggcagtggc
     6781 tcgcgggagg atcggcggga aactccggga tatccgtcga gagtttacac gtgaatgggc
     6841 agcgcaatcc gttgacgacg atacgactgg caaagtagcg acgatacctg ccagacaggt
     6901 gacatgtgca ggccgcacta acaaggaaac gggcgctggg gggggcgggc ttctagactt
//
