LOCUS       sequence-368802-        6663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Parent vector for markerless yeast integration cassettes with 
            N-terminal Neon(gfp)-tagged genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6663)
  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 6663)
  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: pBBK25
            pHomL-TDH3p-Neon(gfp)-Link-GFPdropout-S...
FEATURES             Location/Qualifiers
     source          1..6663
                     /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"
     regulatory      3763..3772
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             4398..4409
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4416..5129
                     /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      5141..5212
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5228..5255
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
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 gtttctaagg gtgaagaaga taacatggct
     3541 tctttgccag ccacccacga gttgcacatc ttcggttcca ttaacggtgt cgacttcgat
     3601 atggtcggtc aaggtaccgg taacccaaac gacggttacg aagaattgaa cttgaagtct
     3661 accaagggcg acttgcagtt ctccccttgg attttggttc cacacatcgg ttacggtttc
     3721 caccaatact tgccataccc agacggtatg tccccattcc aagccgccat ggttgacggt
     3781 tctggttacc aagtccacag aaccatgcaa ttcgaagatg gtgcttcctt gaccgtgaac
     3841 tacagataca cctacgaagg ttctcacatc aagggcgaag cccaagttaa gggtaccggt
     3901 ttccctgctg acggtccagt catgactaac tccttgaccg ctgctgactg gtgtcgttcc
     3961 aagaaaacct acccaaacga taagaccatc atttccactt tcaagtggtc ctacaccacc
     4021 ggtaacggca agagataccg ctctaccgct cgtaccacct acaccttcgc caagccaatg
     4081 gctgccaact acttgaagaa ccagcctatg tacgttttcc gtaagaccga gctgaagcat
     4141 tccaagactg aattgaactt caaggaatgg caaaaggctt tcaccgacgt catgggtatg
     4201 gacgaattgt acaagtctgg tccttctggc ccaactggtt cttgagaccg aaagtgaaac
     4261 gtgatttcat gcgtcatttt gaacattttg taaatcttat ttaataatgt gtgcggcaat
     4321 tcacatttaa tttatgaatg ttttcttaac atcgcggcaa ctcaagaaac ggcaggttcg
     4381 gatcttagct actagagaaa gaggagaaat actagatgcg taaaggcgaa gagctgttca
     4441 ctggtgtcgt ccctattctg gtggaactgg atggtgatgt caacggtcat aagttttccg
     4501 tgcgtggcga gggtgaaggt gacgcaacta atggtaaact gacgctgaag ttcatctgta
     4561 ctactggtaa actgccggtt ccttggccga ctctggtaac gacgctgact tatggtgttc
     4621 agtgctttgc tcgttatccg gaccatatga agcagcatga cttcttcaag tccgccatgc
     4681 cggaaggcta tgtgcaggaa cgcacgattt cctttaagga tgacggcacg tacaaaacgc
     4741 gtgcggaagt gaaatttgaa ggcgataccc tggtaaaccg cattgagctg aaaggcattg
     4801 actttaaaga ggacggcaat atcctgggcc ataagctgga atacaatttt aacagccaca
     4861 atgtttacat caccgccgat aaacaaaaaa atggcattaa agcgaatttt aaaattcgcc
     4921 acaacgtgga ggatggcagc gtgcagctgg ctgatcacta ccagcaaaac actccaatcg
     4981 gtgatggtcc tgttctgctg ccagacaatc actatctgag cacgcaaagc gttctgtcta
     5041 aagatccgaa cgagaaacgc gatcatatgg ttctgctgga gttcgtaacc gcagcgggca
     5101 tcacgcatgg tatggatgaa ctgtacaaat gaccaggcat caaataaaac gaaaggctca
     5161 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag
     5221 tcacactggc tcaccttcgg gtgggccttt ctgcgtttat aggtctcaat cctaactcga
     5281 ggccaattgg tgcggcaatt gataataacg aaaatgtctt ttaatgatct gggtataatg
     5341 aggaattttc cgaacgtttt tactttatat atatatatac atgtaacata tattctatac
     5401 gctatagaga aaggaaattt ttcaattaaa aaaaaaatag agaaagagtt tcacttcttg
     5461 attatcgcta acactaatgg ttgaagtact gctactttaa ttttatgctg ccaatgagac
     5521 gacggggtca tcacggctca tcatgcgcca aacaaatgtg tgcaatacac gctcggatga
     5581 ctgcatgatg accgcactga ctggggacag cagatccacc taagcctgtg agagaagcag
     5641 acacccgaca gatcaaggca gttaactagt gcactgcagt acaaatcgtc cccaacaaaa
     5701 gtgggctctc aaaattcatc acatttaaat gcatatagga agagcaacag ttggtttgca
     5761 tctgatgttc cttaaagatt tcgacataat gtgcgaagta gatcccttca gcactgtact
     5821 tgtttttgcg actagattgt aaatcattct ttatttaatc tctttcttta actactgctt
     5881 aaagtataat ttggtccgta gtttaataac tatactaagc gaacgctctc tcgaaatttt
     5941 tcttaacgcg tccttgtact gcgtctaacg cttttgccac ttggatttct attataggaa
     6001 atagtctcac ttactgggcg acgaattttc gcgttttgat gaagcacagg aagaatttct
     6061 ttatgctcag aagtttgaca gcaagcaagt tcatcattcg aactagcctt attgttttag
     6121 ttcagtgaca gcgaactgcc gtactcgatg ctttatttct cacggtagag cggaagaaca
     6181 gatagatccc tacatattgc taccacttct attacacaat agtttcaata gcttgcagcg
     6241 tagctaaact ctaaaattta tctaaatcac tcatataaac cgaacccttc cccttccgct
     6301 tatagtacag ctgatgctcc ttcactattt taacatgtgg aattcttgaa agaatgaaat
     6361 cgccatgcca agccatcaca cggtctttta tgcaattgat tgaccgcctg caacacatag
     6421 gcagtaaaat ttttacaagc agtagcagaa caggccacac aatcgcaagt gattaacgtc
     6481 cacacaggta tagggtttct ggaccatatg atacatgctc tggccaagca ttccggctgg
     6541 tcgctaatcg ttgagtgccg agcggccgcg attatcaaaa aggatcttca cctagatcct
     6601 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6661 cag
//
