LOCUS       sequence-360753-        6683 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 6683)
  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 6683)
  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: pBBK27
            pHomL-RPL18Bp-Neon(gfp)-Link-GFPdropout...
FEATURES             Location/Qualifiers
     source          1..6683
                     /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"
     regulatory      3783..3792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             4418..4429
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4436..5149
                     /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      5161..5232
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5248..5275
                     /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 gaagaggatg tccaatattt tttttaagga ataaggatac ttcaagacta gattcccccc
     2881 tgcattccca tcagaaccgt aaaccttggc gctttccttg ggaagtattc aagaagtgcc
     2941 ttgtccggtt tctgtggctc acaaaccagc gcgcccgata tggctttctt ttcacttatg
     3001 aatgtaccag tacgggacaa ttagaacgct cctgtaacaa tctctttgca aatgtggggt
     3061 tacattctaa ccatgtcaca ctgctgacga aattcaaagt aaaaaaaaat gggaccacgt
     3121 cttgagaacg atagattttc tttattttac attgaacagt cgttgtctca gcgcgcttta
     3181 tgttttcatt catacttcat attataaaat aacaaaagaa gaatttcata ttcacgccca
     3241 agaaatcagg ctgctttcca aatgcaattg acacttcatt agccatcaca caaaactctt
     3301 tcttgctgga gcttctttta aaaaagacct cagtacacca aacacgttac ccgacctcgt
     3361 tattttacga caactatgat aaaattctga agaaaaaata aaaaaatttt catacttctt
     3421 gcttttattt aaaccattga atgatttctt ttgaacaaaa ctacctgttt caccaaagga
     3481 aatagaaaga aaaaatcaat tagaagaaaa caaaaaacaa aagatctatg gtttctaagg
     3541 gtgaagaaga taacatggct tctttgccag ccacccacga gttgcacatc ttcggttcca
     3601 ttaacggtgt cgacttcgat atggtcggtc aaggtaccgg taacccaaac gacggttacg
     3661 aagaattgaa cttgaagtct accaagggcg acttgcagtt ctccccttgg attttggttc
     3721 cacacatcgg ttacggtttc caccaatact tgccataccc agacggtatg tccccattcc
     3781 aagccgccat ggttgacggt tctggttacc aagtccacag aaccatgcaa ttcgaagatg
     3841 gtgcttcctt gaccgtgaac tacagataca cctacgaagg ttctcacatc aagggcgaag
     3901 cccaagttaa gggtaccggt ttccctgctg acggtccagt catgactaac tccttgaccg
     3961 ctgctgactg gtgtcgttcc aagaaaacct acccaaacga taagaccatc atttccactt
     4021 tcaagtggtc ctacaccacc ggtaacggca agagataccg ctctaccgct cgtaccacct
     4081 acaccttcgc caagccaatg gctgccaact acttgaagaa ccagcctatg tacgttttcc
     4141 gtaagaccga gctgaagcat tccaagactg aattgaactt caaggaatgg caaaaggctt
     4201 tcaccgacgt catgggtatg gacgaattgt acaagtctgg tccttctggc ccaactggtt
     4261 cttgagaccg aaagtgaaac gtgatttcat gcgtcatttt gaacattttg taaatcttat
     4321 ttaataatgt gtgcggcaat tcacatttaa tttatgaatg ttttcttaac atcgcggcaa
     4381 ctcaagaaac ggcaggttcg gatcttagct actagagaaa gaggagaaat actagatgcg
     4441 taaaggcgaa gagctgttca ctggtgtcgt ccctattctg gtggaactgg atggtgatgt
     4501 caacggtcat aagttttccg tgcgtggcga gggtgaaggt gacgcaacta atggtaaact
     4561 gacgctgaag ttcatctgta ctactggtaa actgccggtt ccttggccga ctctggtaac
     4621 gacgctgact tatggtgttc agtgctttgc tcgttatccg gaccatatga agcagcatga
     4681 cttcttcaag tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt cctttaagga
     4741 tgacggcacg tacaaaacgc gtgcggaagt gaaatttgaa ggcgataccc tggtaaaccg
     4801 cattgagctg aaaggcattg actttaaaga ggacggcaat atcctgggcc ataagctgga
     4861 atacaatttt aacagccaca atgtttacat caccgccgat aaacaaaaaa atggcattaa
     4921 agcgaatttt aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg ctgatcacta
     4981 ccagcaaaac actccaatcg gtgatggtcc tgttctgctg ccagacaatc actatctgag
     5041 cacgcaaagc gttctgtcta aagatccgaa cgagaaacgc gatcatatgg ttctgctgga
     5101 gttcgtaacc gcagcgggca tcacgcatgg tatggatgaa ctgtacaaat gaccaggcat
     5161 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     5221 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
     5281 aggtctcaat cctaactcga ggccaattgg tgcggcaatt gataataacg aaaatgtctt
     5341 ttaatgatct gggtataatg aggaattttc cgaacgtttt tactttatat atatatatac
     5401 atgtaacata tattctatac gctatagaga aaggaaattt ttcaattaaa aaaaaaatag
     5461 agaaagagtt tcacttcttg attatcgcta acactaatgg ttgaagtact gctactttaa
     5521 ttttatgctg ccaatgagac gacggggtca tcacggctca tcatgcgcca aacaaatgtg
     5581 tgcaatacac gctcggatga ctgcatgatg accgcactga ctggggacag cagatccacc
     5641 taagcctgtg agagaagcag acacccgaca gatcaaggca gttaactagt gcactgcagt
     5701 acaaatcgtc cccaacaaaa gtgggctctc aaaattcatc acatttaaat gcatatagga
     5761 agagcaacag ttggtttgca tctgatgttc cttaaagatt tcgacataat gtgcgaagta
     5821 gatcccttca gcactgtact tgtttttgcg actagattgt aaatcattct ttatttaatc
     5881 tctttcttta actactgctt aaagtataat ttggtccgta gtttaataac tatactaagc
     5941 gaacgctctc tcgaaatttt tcttaacgcg tccttgtact gcgtctaacg cttttgccac
     6001 ttggatttct attataggaa atagtctcac ttactgggcg acgaattttc gcgttttgat
     6061 gaagcacagg aagaatttct ttatgctcag aagtttgaca gcaagcaagt tcatcattcg
     6121 aactagcctt attgttttag ttcagtgaca gcgaactgcc gtactcgatg ctttatttct
     6181 cacggtagag cggaagaaca gatagatccc tacatattgc taccacttct attacacaat
     6241 agtttcaata gcttgcagcg tagctaaact ctaaaattta tctaaatcac tcatataaac
     6301 cgaacccttc cccttccgct tatagtacag ctgatgctcc ttcactattt taacatgtgg
     6361 aattcttgaa agaatgaaat cgccatgcca agccatcaca cggtctttta tgcaattgat
     6421 tgaccgcctg caacacatag gcagtaaaat ttttacaagc agtagcagaa caggccacac
     6481 aatcgcaagt gattaacgtc cacacaggta tagggtttct ggaccatatg atacatgctc
     6541 tggccaagca ttccggctgg tcgctaatcg ttgagtgccg agcggccgcg attatcaaaa
     6601 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6661 tatgagtaaa cttggtctga cag
//
