LOCUS       sequence-368804-        6664 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Parent vector for markerless yeast integration cassettes with 
            C-terminal Neon(gfp)-tagged genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6664)
  AUTHORS   Bean BDM, Whiteway M, Martin VJJ
  TITLE     The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and 
            Overexpression CRISPR-Cas9 Toolkit.
  JOURNAL   G3 (Bethesda). 2022 Jun 16. pii: 6609175. doi: 
            10.1093/g3journal/jkac154.
  PUBMED    35708612
REFERENCE   2  (bases 1 to 6664)
  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: "G3 
            (Bethesda). 2022 Jun 16. pii: 6609175. doi: 
            10.1093/g3journal/jkac154."
COMMENT     Sequence Label: pBBK28
            pHomL-TDH3p-GFPdropout-Link-Neon(gfp)-S...
FEATURES             Location/Qualifiers
     source          1..6664
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1069..1657
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1558..1577
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2835..3501
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3371..3394
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     RBS             3666..3677
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3684..4397
                     /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      4409..4480
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4496..4523
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     regulatory      4817..4826
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gggacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgtcatgacc aaaatccctt aacgtgagtt
     1021 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     1081 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     1141 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     1201 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     1261 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     1321 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     1381 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     1441 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     1501 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     1561 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     1621 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     1681 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     1741 ttctgtggat aaccgtgcgg ccgccaatgt tatctattgg cagatgttca tacggggctt
     1801 tcaaatattg atgaaattat gtgatgttta gaagaagatt cgaactgttt tcagtagatt
     1861 tggtaactgt gcaaccataa ctcatgcccc ctatttatcg gcattgagat tgtaaaggaa
     1921 gtaaaacgca tttttgcaga tctgttctct tatgtatttt taatcgtcct tgtatggaag
     1981 tatcaaaggg gacgttcttc acctccttgg aaaacgcaaa ctttcttttt gcctatcaca
     2041 gtatcttatc gagctaacta ttttcgacac acatgaaaaa gcagaaatat taacgaaaaa
     2101 gaaaagaaag accatgtcat gtacgggcaa tcagaatatg caagatgagc gggaacgggc
     2161 gaaatgtgca cgcgcacaac ttacgaaacg cggatgagtc actgacagcc accgcagagg
     2221 ttctgactcc tactgagctc tattggaggt ggcagaaccg gtaatcccac agtgagcaac
     2281 ttgacaaacc aacagatata ggctgtgtct taaaatatac catccaatac cttgatgaac
     2341 ttttcaaaga tgactaaaat aagtgaaatt tcaacattaa cttcgagctg taaacacagt
     2401 ggtttctttg cataaacacc atcagcctca agtcgtcaag taaagatttc gtgttcatgc
     2461 agatagataa caatctatat gttgataatt agcgttgcct catcaatgcg tacacgaaga
     2521 atatactaaa aaatgagcag gcaagataaa cgaaggcaaa gatgacagag cagaaagccc
     2581 tagtaaagcg tattacaaat gaaaccaaga ttcagattgc gatccctgaa ttcgcatcta
     2641 gatggtagag ccacaaacag ccggtacaag caacgatctc caggaccatc tgaatcatgc
     2701 gcggatgaca cgaactcacg acggcgatca cagacattaa cccacagtac agacactgcg
     2761 acaacgtggc aattcgtcgc aataccgtct cactgaactg gccgataatt gcagacgaac
     2821 gcagttcgag tttatcatta tcaatactgc catttcaaag aatacgtaaa taattaatag
     2881 tagtgatttt cctaacttta tttagtcaaa aaattagcct tttaattctg ctgtaacccg
     2941 tacatgccca aaataggggg cgggttacac agaatatata acatcgtagg tgtctgggtg
     3001 aacagtttat tcctggcatc cactaaatat aatggagccc gctttttaag ctggcatcca
     3061 gaaaaaaaaa gaatcccagc accaaaatat tgttttcttc accaaccatc agttcatagg
     3121 tccattctct tagcgcaact acagagaaca ggggcacaaa caggcaaaaa acgggcacaa
     3181 cctcaatgga gtgatgcaac ctgcctggag taaatgatga cacaaggcaa ttgacccacg
     3241 catgtatcta tctcattttc ttacaccttc tattaccttc tgctctctct gatttggaaa
     3301 aagctgaaaa aaaaggttga aaccagttcc ctgaaattat tcccctactt gactaataag
     3361 tatataaaga cggtaggtat tgattgtaat tctgtaaatc tatttcttaa acttcttaaa
     3421 ttctactttt atagttagtc ttttttttag ttttaaaaca ccaagaactt agtttcgaat
     3481 aaacacacat aaacaaacaa aagatctatg tgagaccgaa agtgaaacgt gatttcatgc
     3541 gtcattttga acattttgta aatcttattt aataatgtgt gcggcaattc acatttaatt
     3601 tatgaatgtt ttcttaacat cgcggcaact caagaaacgg caggttcgga tcttagctac
     3661 tagagaaaga ggagaaatac tagatgcgta aaggcgaaga gctgttcact ggtgtcgtcc
     3721 ctattctggt ggaactggat ggtgatgtca acggtcataa gttttccgtg cgtggcgagg
     3781 gtgaaggtga cgcaactaat ggtaaactga cgctgaagtt catctgtact actggtaaac
     3841 tgccggttcc ttggccgact ctggtaacga cgctgactta tggtgttcag tgctttgctc
     3901 gttatccgga ccatatgaag cagcatgact tcttcaagtc cgccatgccg gaaggctatg
     3961 tgcaggaacg cacgatttcc tttaaggatg acggcacgta caaaacgcgt gcggaagtga
     4021 aatttgaagg cgataccctg gtaaaccgca ttgagctgaa aggcattgac tttaaagagg
     4081 acggcaatat cctgggccat aagctggaat acaattttaa cagccacaat gtttacatca
     4141 ccgccgataa acaaaaaaat ggcattaaag cgaattttaa aattcgccac aacgtggagg
     4201 atggcagcgt gcagctggct gatcactacc agcaaaacac tccaatcggt gatggtcctg
     4261 ttctgctgcc agacaatcac tatctgagca cgcaaagcgt tctgtctaaa gatccgaacg
     4321 agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc acgcatggta
     4381 tggatgaact gtacaaatga ccaggcatca aataaaacga aaggctcagt cgaaagactg
     4441 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc
     4501 accttcgggt gggcctttct gcgtttatag gtctcaatcc ggtccttctg gcccaactgg
     4561 catggtttct aagggtgaag aagataacat ggcttctttg ccagccaccc acgagttgca
     4621 catcttcggt tccattaacg gtgtcgactt cgatatggtc ggtcaaggta ccggtaaccc
     4681 aaacgacggt tacgaagaat tgaacttgaa gtctaccaag ggcgacttgc agttctcccc
     4741 ttggattttg gttccacaca tcggttacgg tttccaccaa tacttgccat acccagacgg
     4801 tatgtcccca ttccaagccg ccatggttga cggttctggt taccaagtcc acagaaccat
     4861 gcaattcgaa gatggtgctt ccttgaccgt gaactacaga tacacctacg aaggttctca
     4921 catcaagggc gaagcccaag ttaagggtac cggtttccct gctgacggtc cagtcatgac
     4981 taactccttg accgctgctg actggtgtcg ttccaagaaa acctacccaa acgataagac
     5041 catcatttcc actttcaagt ggtcctacac caccggtaac ggcaagagat accgctctac
     5101 cgctcgtacc acctacacct tcgccaagcc aatggctgcc aactacttga agaaccagcc
     5161 tatgtacgtt ttccgtaaga ccgagctgaa gcattccaag actgaattga acttcaagga
     5221 atggcaaaag gctttcaccg acgtcatggg tatggacgaa ttgtacaagt aactcgagtg
     5281 gcgccaattg gtgcggcaat tgataataac gaaaatgtct tttaatgatc tgggtataat
     5341 gaggaatttt ccgaacgttt ttactttata tatatatata catgtaacat atattctata
     5401 cgctatagag aaaggaaatt tttcaattaa aaaaaaaata gagaaagagt ttcacttctt
     5461 gattatcgct aacactaatg gttgaagtac tgctacttta attttatgct gccaatgaga
     5521 cgacggggtc atcacggctc atcatgcgcc aaacaaatgt gtgcaataca cgctcggatg
     5581 actgcatgat gaccgcactg actggggaca gcagatccac ctaagcctgt gagagaagca
     5641 gacacccgac agatcaaggc agttaactag tgcactgcag tacaaatcgt ccccaacaaa
     5701 agtgggctct caaaattcat cacatttaaa tgcatatagg aagagcaaca gttggtttgc
     5761 atctgatgtt ccttaaagat ttcgacataa tgtgcgaagt agatcccttc agcactgtac
     5821 ttgtttttgc gactagattg taaatcattc tttatttaat ctctttcttt aactactgct
     5881 taaagtataa tttggtccgt agtttaataa ctatactaag cgaacgctct ctcgaaattt
     5941 ttcttaacgc gtccttgtac tgcgtctaac gcttttgcca cttggatttc tattatagga
     6001 aatagtctca cttactgggc gacgaatttt cgcgttttga tgaagcacag gaagaatttc
     6061 tttatgctca gaagtttgac agcaagcaag ttcatcattc gaactagcct tattgtttta
     6121 gttcagtgac agcgaactgc cgtactcgat gctttatttc tcacggtaga gcggaagaac
     6181 agatagatcc ctacatattg ctaccacttc tattacacaa tagtttcaat agcttgcagc
     6241 gtagctaaac tctaaaattt atctaaatca ctcatataaa ccgaaccctt ccccttccgc
     6301 ttatagtaca gctgatgctc cttcactatt ttaacatgtg gaattcttga aagaatgaaa
     6361 tcgccatgcc aagccatcac acggtctttt atgcaattga ttgaccgcct gcaacacata
     6421 ggcagtaaaa tttttacaag cagtagcaga acaggccaca caatcgcaag tgattaacgt
     6481 ccacacaggt atagggtttc tggaccatat gatacatgct ctggccaagc attccggctg
     6541 gtcgctaatc gttgagtgcc gagcggccgc gattatcaaa aaggatcttc acctagatcc
     6601 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6661 acag
//
