LOCUS       sequence-424077-        4803 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integration vector for three gene cassette with integration site of 
            the gene 4HPPD locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4803)
  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 4803)
  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: pCfB13017
FEATURES             Location/Qualifiers
     source          1..4803
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      340..411
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      427..454
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    complement(923..956)
                     /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(1163..1182)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1188..1207)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1249..1340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1341..1409,1410..2201)
                     /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(1559..1578)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2323..2911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2812..2831
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3065..3082
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3180..3197
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3181..3197
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3667..3700
                     /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)"
     RBS             4400..4411
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4418..4803,1..328)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
ORIGIN
        1 ctttaaagag gacggcaata tcctgggcca taagctggaa tacaatttta acagccacaa
       61 tgtttacatc accgccgata aacaaaaaaa tggcattaaa gcgaatttta aaattcgcca
      121 caacgtggag gatggcagcg tgcagctggc tgatcactac cagcaaaaca ctccaatcgg
      181 tgatggtcct gttctgctgc cagacaatca ctatctgagc acgcaaagcg ttctgtctaa
      241 agatccgaac gagaaacgcg atcatatggt tctgctggag ttcgtaaccg cagcgggcat
      301 cacgcatggt atggatgaac tgtacaaatg accaggcatc aaataaaacg aaaggctcag
      361 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct ctactagagt
      421 cacactggct caccttcggg tgggcctttc tgcgtttata cgtctcagtt cggtctcagc
      481 tggttgttgg tggttatttg tttgtgtgtc aaaattgtgt agagagagaa aattctaggt
      541 cgagcccatc ttctatatat attggagatt cttatttgtg tcgcatacgc tctgaaaaaa
      601 aaaaaaggtc caaaatgcac attaagatta tcgcggtgca caatcagtct ccgcgtctta
      661 ccggaggggt accaaacggc gcaaagttat cggctgttag ggttcatcgt agtggaggag
      721 caagtgtctc gtgaggaggg gtagaggggg ggtttacggt agtggcaaac attggaaatt
      781 gtgcccccca aacaacacat tttcccactc cttctctgta ccatctcaca cccattgccg
      841 aattgcacac tagtgcaaca agttataata ccacccacca cctccaatac ccgggggcgg
      901 ccgcatttaa atccccactt cagaagttcc tatactttct agagaatagg aacttcacta
      961 tagagtcgaa taagggcgac accccataat tagcccgggc gaaaggccca gtctttcgac
     1021 tgagcctttc gttttatttg atgcctggca gttccctact ctcgcatggg gagtccccac
     1081 actaccatcg gcgctacggc gtttcacttc tgagttcggc atggggtcag gtgggaccac
     1141 cgcgctactg ccgccaggca aacaaggggt gttatgagcc atattcaggt ataaatgggc
     1201 tcgcgataat gttcagaatt ggttaattgg ttgtaacact gacccctatt tgtttatttt
     1261 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1321 aatattgaaa aaggaagaat atgagtattc aacatttccg tgtcgccctt attccctttt
     1381 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1441 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1501 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1561 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     1621 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     1681 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     1741 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     1801 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     1861 acgagcgtga caccacgatg cctgtagcga tggcaacaac gttgcgcaaa ctattaactg
     1921 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     1981 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatccg
     2041 gagccggtga gcgtggttct cgcggtatca tcgcagcgct ggggccagat ggtaagccct
     2101 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2161 agatcgctga gataggtgcc tcactgatta agcattggta agcagagcat tacgctgact
     2221 tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc gcgcgtcgtt
     2281 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2341 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2401 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2461 aaatactgtt cttctagtgt agccgtagtt agcccaccac ttcaagaact ctgtagcacc
     2521 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2581 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2641 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2701 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2761 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2821 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2881 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2941 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3001 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3061 gcgcagcgag tcagtgagcg aggaagcgga aggcgagagt agggaactgc caggcatcaa
     3121 actaagcaga aggcccctga cggatggcct ttttgcgttt ctacaaactc tttctgtgtt
     3181 gtaaaacgac ggccagtctt aagctcgggc cccctgggcg gttctgataa cgagtaatcg
     3241 ttaatccgca aataacgtaa aaacccgctt cggcgggttt ttttatgggg ggagtttagg
     3301 gaaagagcat ttgtcagaat atttaagggc gcctgtcact ttgcttgata tatgagaatt
     3361 atttaacctt ataaatgaga aaaaagcaac gcactttaaa taagatacgt tgctttttcg
     3421 attgatgaac acctataatt aaactattca tctattattt atgatttttt gtatatacaa
     3481 tatttctagt ttgttaaaga gaattaagaa aataaatctc gaaaataata aagggaaaat
     3541 cagtttttga tatcaaaatt atacatgtca acgataatac aaaatataat acaaactata
     3601 agatgttatc agtatttatt atgcatttag aataaatttt gtgtcgccct tattcgactc
     3661 actatagaag ttcctattct ctagaaagta taggaacttc acttcatttt atttaaattt
     3721 gcggccgcca gcggacatag ccggcgatga aaaccaatcg ttgcggacaa attaagcacc
     3781 caataaatga cttgaaaccg tggctaaaac gcaggaacct cagccgttca aaagtcgctt
     3841 caaatacaca gtctacacac cttccacagt aggtaaatac tctacccttc attggacacc
     3901 tcctctttaa gctaacctct tataatatat actacggaga acaatcaaaa attagcctgg
     3961 tccccttcct cggctaaaac ttcaatggag caactccaac ccgataatct gcccattctc
     4021 cggagaattc tcttccggca cttggcacaa tgagcgcgct ttacaaacgc agtttcatgc
     4081 ggcgggtgtg gcgagcgtgg cgcgtatcag ctttatgatg atctacaggt atgtactaca
     4141 agtacctact gctacctgga ataccacgta gtatacaagt ttgctcatat ttatctgtgt
     4201 attgcttggc caggttaatt gaaattgcaa cgtgagacca ctgatgagac ggaaagtgaa
     4261 acgtgatttc atgcgtcatt ttgaacattt tgtaaatctt atttaataat gtgtgcggca
     4321 attcacattt aatttatgaa tgttttctta acatcgcggc aactcaagaa acggcaggtt
     4381 cggatcttag ctactagaga aagaggagaa atactagatg cgtaaaggcg aagagctgtt
     4441 cactggtgtc gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc
     4501 cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg
     4561 tactactggt aaactgccgg ttccttggcc gactctggta acgacgctga cttatggtgt
     4621 tcagtgcttt gctcgttatc cggaccatat gaagcagcat gacttcttca agtccgccat
     4681 gccggaaggc tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac
     4741 gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat
     4801 tga
//
