LOCUS       sequence-424256-        4872 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integration vector for three gene cassette with integration site F3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4872)
  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 4872)
  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: pCfB12718
FEATURES             Location/Qualifiers
     source          1..4872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             229..240
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             247..960
                     /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"
     terminator      972..1043
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1059..1086
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    complement(1656..1689)
                     /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(1896..1915)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1921..1940)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1982..2073
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2074..2142,2143..2934)
                     /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(2292..2311)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3056..3644
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3545..3564
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3798..3815
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3913..3930
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3914..3930
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4400..4433
                     /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)"
ORIGIN
        1 attcaccgaa ctatccttga gatactcatt gctcatcgct acgggctcga cgcacggaac
       61 gtgagaccac tgatgagacg gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt
      121 gtaaatctta tttaataatg tgtgcggcaa ttcacattta atttatgaat gttttcttaa
      181 catcgcggca actcaagaaa cggcaggttc ggatcttagc tactagagaa agaggagaaa
      241 tactagatgc gtaaaggcga agagctgttc actggtgtcg tccctattct ggtggaactg
      301 gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg tgacgcaact
      361 aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt tccttggccg
      421 actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg
      481 aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga acgcacgatt
      541 tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc
      601 ctggtaaacc gcattgagct gaaaggcatt gactttaaag aggacggcaa tatcctgggc
      661 cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga taaacaaaaa
      721 aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag cgtgcagctg
      781 gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct gccagacaat
      841 cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg cgatcatatg
      901 gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga actgtacaaa
      961 tgaccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     1021 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
     1081 tctgcgttta tacgtctcag ttcggtctca gctggagtga gtttctgggg accgagaatt
     1141 agtgttgatg ctagcgcaag gcttgttctt tagtgggcat ctaccgcagc tactatacac
     1201 gagcgcgacg taggtatagc atgccaaagg cgggtgcgat accagatgac gctataatat
     1261 gccagatttg cactagcaca catcactgga agctgtcaat ttgaaaatgt caatgcagtc
     1321 tcaccattgg acgatcttgg cacctcctaa actctggcaa tgtctgagct ctttgacaca
     1381 taagacatca agcagacggt ctaaaatcat atgccatgta aagaactact agtgcctgta
     1441 gaagacgcct gtagaagacg cctgtagaag acgcctgtag aagacggcta cttgtatatt
     1501 ctcacttgtc gtgcttcact cacggctcga tgtccactgc ttacctcagc ggcagcgggc
     1561 ggaaaaagcc tacaaacccc tgtcggccgc gttgcacttg ttgggcgacg accaaaatcg
     1621 cgtgcattcg cggccgcatt taaatcccca cttcagaagt tcctatactt tctagagaat
     1681 aggaacttca ctatagagtc gaataagggc gacaccccat aattagcccg ggcgaaaggc
     1741 ccagtctttc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcat
     1801 ggggagtccc cacactacca tcggcgctac ggcgtttcac ttctgagttc ggcatggggt
     1861 caggtgggac caccgcgcta ctgccgccag gcaaacaagg ggtgttatga gccatattca
     1921 ggtataaatg ggctcgcgat aatgttcaga attggttaat tggttgtaac actgacccct
     1981 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2041 taaatgcttc aataatattg aaaaaggaag aatatgagta ttcaacattt ccgtgtcgcc
     2101 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2161 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2221 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2281 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2341 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2401 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2461 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2521 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2581 gccataccaa acgacgagcg tgacaccacg atgcctgtag cgatggcaac aacgttgcgc
     2641 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2701 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2761 gctgataaat ccggagccgg tgagcgtggt tctcgcggta tcatcgcagc gctggggcca
     2821 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2881 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaagcagag
     2941 cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagtta
     3001 cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3061 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3121 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3181 gagcgcagat accaaatact gttcttctag tgtagccgta gttagcccac cacttcaaga
     3241 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3301 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3361 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3421 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3481 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3541 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3601 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3661 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3721 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3781 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaggcgag agtagggaac
     3841 tgccaggcat caaactaagc agaaggcccc tgacggatgg cctttttgcg tttctacaaa
     3901 ctctttctgt gttgtaaaac gacggccagt cttaagctcg ggccccctgg gcggttctga
     3961 taacgagtaa tcgttaatcc gcaaataacg taaaaacccg cttcggcggg tttttttatg
     4021 gggggagttt agggaaagag catttgtcag aatatttaag ggcgcctgtc actttgcttg
     4081 atatatgaga attatttaac cttataaatg agaaaaaagc aacgcacttt aaataagata
     4141 cgttgctttt tcgattgatg aacacctata attaaactat tcatctatta tttatgattt
     4201 tttgtatata caatatttct agtttgttaa agagaattaa gaaaataaat ctcgaaaata
     4261 ataaagggaa aatcagtttt tgatatcaaa attatacatg tcaacgataa tacaaaatat
     4321 aatacaaact ataagatgtt atcagtattt attatgcatt tagaataaat tttgtgtcgc
     4381 ccttattcga ctcactatag aagttcctat tctctagaaa gtataggaac ttcacttcat
     4441 tttatttaaa tttgcggccg cgaatgcgtg cgatgcccat caatccatgg aatctttgcc
     4501 gtgagacaca caagtttcag acttgctcct tttgagtctt ctcatcattt acgacagttc
     4561 tttttccacc cacagccatc cgacttgtgc atctccgtat cctagccgag cccattcggg
     4621 gtgcatgaca actgcgggat cttgggggta gagtgtttag cgaaattaat atatgcagta
     4681 tagtgcagga tgagctaaga agcattctac aaacagttgt atacaggcac cagacattta
     4741 tatagttatt gggaaacgtc gagtggcgta agatggcggt gccctggagc cactcacaca
     4801 tgtatcatag gtttaataag tcccgcacgg gtcgtttcta atgtaatgtc tcatgtctaa
     4861 gaaccacggt gt
//
