LOCUS       sequence-360776-        6054 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Parent vector for markerless yeast integration cassettes with 
            C-terminal 3xHemagglutinin-tagged genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6054)
  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 6054)
  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: pBBK42 pHomL-RPL18Bp-GFPdropout-Link-3HA-SSA1t...
FEATURES             Location/Qualifiers
     source          1..6054
                     /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"
     CDS             4573..4599
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4603..4629
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4633..4659
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
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 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 ccttctggca tgtaccccta cgacgttccc gactacgctt cttatccata tgatgtgcca
     4621 gattacgcgg gatatccgta tgatgtcccg gattacgcct aactcgagtg gcgccaattg
     4681 gtgcggcaat tgataataac gaaaatgtct tttaatgatc tgggtataat gaggaatttt
     4741 ccgaacgttt ttactttata tatatatata catgtaacat atattctata cgctatagag
     4801 aaaggaaatt tttcaattaa aaaaaaaata gagaaagagt ttcacttctt gattatcgct
     4861 aacactaatg gttgaagtac tgctacttta attttatgct gccaatgaga cgacggggtc
     4921 atcacggctc atcatgcgcc aaacaaatgt gtgcaataca cgctcggatg actgcatgat
     4981 gaccgcactg actggggaca gcagatccac ctaagcctgt gagagaagca gacacccgac
     5041 agatcaaggc agttaactag tgcactgcag tacaaatcgt ccccaacaaa agtgggctct
     5101 caaaattcat cacatttaaa tgcatatagg aagagcaaca gttggtttgc atctgatgtt
     5161 ccttaaagat ttcgacataa tgtgcgaagt agatcccttc agcactgtac ttgtttttgc
     5221 gactagattg taaatcattc tttatttaat ctctttcttt aactactgct taaagtataa
     5281 tttggtccgt agtttaataa ctatactaag cgaacgctct ctcgaaattt ttcttaacgc
     5341 gtccttgtac tgcgtctaac gcttttgcca cttggatttc tattatagga aatagtctca
     5401 cttactgggc gacgaatttt cgcgttttga tgaagcacag gaagaatttc tttatgctca
     5461 gaagtttgac agcaagcaag ttcatcattc gaactagcct tattgtttta gttcagtgac
     5521 agcgaactgc cgtactcgat gctttatttc tcacggtaga gcggaagaac agatagatcc
     5581 ctacatattg ctaccacttc tattacacaa tagtttcaat agcttgcagc gtagctaaac
     5641 tctaaaattt atctaaatca ctcatataaa ccgaaccctt ccccttccgc ttatagtaca
     5701 gctgatgctc cttcactatt ttaacatgtg gaattcttga aagaatgaaa tcgccatgcc
     5761 aagccatcac acggtctttt atgcaattga ttgaccgcct gcaacacata ggcagtaaaa
     5821 tttttacaag cagtagcaga acaggccaca caatcgcaag tgattaacgt ccacacaggt
     5881 atagggtttc tggaccatat gatacatgct ctggccaagc attccggctg gtcgctaatc
     5941 gttgagtgcc gagcggccgc gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6001 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acag
//
