LOCUS       sequence-360758-        6684 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 6684)
  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 6684)
  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: pBBK29
            pHomL-TEF2p-GFPdropout-Link-Neon(gfp)-S...
FEATURES             Location/Qualifiers
     source          1..6684
                     /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"
     RBS             3686..3697
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3704..4417
                     /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      4429..4500
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4516..4543
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     regulatory      4837..4846
                     /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 gttgataggt caagatcaat gtaaacaatt actttgttat gtagagtttt tttagctacc
     2881 tatattccac cataacatca atcatgcggt tgctggtgta tttaccaata atgtttaatg
     2941 tatatatata tatatatata tggggccgta tacttacata tagtagatgt caagcgtagg
     3001 cgcttcccct gccggctgtg agggcgccat aaccaaggta tctatagacc gccaatcagc
     3061 aaactacctc cgtacattca tgttgcaccc acacatttat acacccagac cgcgacaaat
     3121 tacccataag gttgtttgtg acggcgtcgt acaagagaac gtgggaactt tttaggctca
     3181 ccaaaaaaga aagaaaaaat acgagttgct gacagaagcc tcaagaaaaa aaaaattctt
     3241 cttcgactat gctggaggca gagatgatcg agccggtagt taactatata tagctaaatt
     3301 ggttccatca ccttcttttc tggtgtcgct ccttctagtg ctatttctgg cttttcctat
     3361 tttttttttt ccatttttct ttctctcttt ctaatatata aattctcttg cattttctat
     3421 ttttctctct atctattcta cttgtttatt cccttcaagg ttttttttta aggagtactt
     3481 gtttttagaa tatacggtca acgaactata attaactaaa cagatctatg tgagaccgaa
     3541 agtgaaacgt gatttcatgc gtcattttga acattttgta aatcttattt aataatgtgt
     3601 gcggcaattc acatttaatt tatgaatgtt ttcttaacat cgcggcaact caagaaacgg
     3661 caggttcgga tcttagctac tagagaaaga ggagaaatac tagatgcgta aaggcgaaga
     3721 gctgttcact ggtgtcgtcc ctattctggt ggaactggat ggtgatgtca acggtcataa
     3781 gttttccgtg cgtggcgagg gtgaaggtga cgcaactaat ggtaaactga cgctgaagtt
     3841 catctgtact actggtaaac tgccggttcc ttggccgact ctggtaacga cgctgactta
     3901 tggtgttcag tgctttgctc gttatccgga ccatatgaag cagcatgact tcttcaagtc
     3961 cgccatgccg gaaggctatg tgcaggaacg cacgatttcc tttaaggatg acggcacgta
     4021 caaaacgcgt gcggaagtga aatttgaagg cgataccctg gtaaaccgca ttgagctgaa
     4081 aggcattgac tttaaagagg acggcaatat cctgggccat aagctggaat acaattttaa
     4141 cagccacaat gtttacatca ccgccgataa acaaaaaaat ggcattaaag cgaattttaa
     4201 aattcgccac aacgtggagg atggcagcgt gcagctggct gatcactacc agcaaaacac
     4261 tccaatcggt gatggtcctg ttctgctgcc agacaatcac tatctgagca cgcaaagcgt
     4321 tctgtctaaa gatccgaacg agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc
     4381 agcgggcatc acgcatggta tggatgaact gtacaaatga ccaggcatca aataaaacga
     4441 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     4501 tactagagtc acactggctc accttcgggt gggcctttct gcgtttatag gtctcaatcc
     4561 ggtccttctg gcccaactgg catggtttct aagggtgaag aagataacat ggcttctttg
     4621 ccagccaccc acgagttgca catcttcggt tccattaacg gtgtcgactt cgatatggtc
     4681 ggtcaaggta ccggtaaccc aaacgacggt tacgaagaat tgaacttgaa gtctaccaag
     4741 ggcgacttgc agttctcccc ttggattttg gttccacaca tcggttacgg tttccaccaa
     4801 tacttgccat acccagacgg tatgtcccca ttccaagccg ccatggttga cggttctggt
     4861 taccaagtcc acagaaccat gcaattcgaa gatggtgctt ccttgaccgt gaactacaga
     4921 tacacctacg aaggttctca catcaagggc gaagcccaag ttaagggtac cggtttccct
     4981 gctgacggtc cagtcatgac taactccttg accgctgctg actggtgtcg ttccaagaaa
     5041 acctacccaa acgataagac catcatttcc actttcaagt ggtcctacac caccggtaac
     5101 ggcaagagat accgctctac cgctcgtacc acctacacct tcgccaagcc aatggctgcc
     5161 aactacttga agaaccagcc tatgtacgtt ttccgtaaga ccgagctgaa gcattccaag
     5221 actgaattga acttcaagga atggcaaaag gctttcaccg acgtcatggg tatggacgaa
     5281 ttgtacaagt aactcgagtg gcgccaattg gtgcggcaat tgataataac gaaaatgtct
     5341 tttaatgatc tgggtataat gaggaatttt ccgaacgttt ttactttata tatatatata
     5401 catgtaacat atattctata cgctatagag aaaggaaatt tttcaattaa aaaaaaaata
     5461 gagaaagagt ttcacttctt gattatcgct aacactaatg gttgaagtac tgctacttta
     5521 attttatgct gccaatgaga cgacggggtc atcacggctc atcatgcgcc aaacaaatgt
     5581 gtgcaataca cgctcggatg actgcatgat gaccgcactg actggggaca gcagatccac
     5641 ctaagcctgt gagagaagca gacacccgac agatcaaggc agttaactag tgcactgcag
     5701 tacaaatcgt ccccaacaaa agtgggctct caaaattcat cacatttaaa tgcatatagg
     5761 aagagcaaca gttggtttgc atctgatgtt ccttaaagat ttcgacataa tgtgcgaagt
     5821 agatcccttc agcactgtac ttgtttttgc gactagattg taaatcattc tttatttaat
     5881 ctctttcttt aactactgct taaagtataa tttggtccgt agtttaataa ctatactaag
     5941 cgaacgctct ctcgaaattt ttcttaacgc gtccttgtac tgcgtctaac gcttttgcca
     6001 cttggatttc tattatagga aatagtctca cttactgggc gacgaatttt cgcgttttga
     6061 tgaagcacag gaagaatttc tttatgctca gaagtttgac agcaagcaag ttcatcattc
     6121 gaactagcct tattgtttta gttcagtgac agcgaactgc cgtactcgat gctttatttc
     6181 tcacggtaga gcggaagaac agatagatcc ctacatattg ctaccacttc tattacacaa
     6241 tagtttcaat agcttgcagc gtagctaaac tctaaaattt atctaaatca ctcatataaa
     6301 ccgaaccctt ccccttccgc ttatagtaca gctgatgctc cttcactatt ttaacatgtg
     6361 gaattcttga aagaatgaaa tcgccatgcc aagccatcac acggtctttt atgcaattga
     6421 ttgaccgcct gcaacacata ggcagtaaaa tttttacaag cagtagcaga acaggccaca
     6481 caatcgcaag tgattaacgt ccacacaggt atagggtttc tggaccatat gatacatgct
     6541 ctggccaagc attccggctg gtcgctaatc gttgagtgcc gagcggccgc gattatcaaa
     6601 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6661 atatgagtaa acttggtctg acag
//
