LOCUS       sequence-360772-        6086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Parent vector for markerless yeast integration cassettes with 
            N-terminal 3xHemagglutinin-tagged genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6086)
  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 6086)
  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: pBBK39 pHomL-RPL18Bp-3HA-Link-GFPdropout-SSA1t...
FEATURES             Location/Qualifiers
     source          1..6086
                     /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"
     CDS             3534..3560
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3567..3593
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3600..3626
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     RBS             3821..3832
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3839..4552
                     /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      4564..4635
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4651..4678
                     /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 gagtacccct
     3541 acgacgttcc cgactacgct gcagagtacc catacgacgt gccagactac gcggcagaat
     3601 atccgtatga tgtcccagat tacgccgcta agttagaatc tggtccttct ggcccaactg
     3661 gttcttgaga ccgaaagtga aacgtgattt catgcgtcat tttgaacatt ttgtaaatct
     3721 tatttaataa tgtgtgcggc aattcacatt taatttatga atgttttctt aacatcgcgg
     3781 caactcaaga aacggcaggt tcggatctta gctactagag aaagaggaga aatactagat
     3841 gcgtaaaggc gaagagctgt tcactggtgt cgtccctatt ctggtggaac tggatggtga
     3901 tgtcaacggt cataagtttt ccgtgcgtgg cgagggtgaa ggtgacgcaa ctaatggtaa
     3961 actgacgctg aagttcatct gtactactgg taaactgccg gttccttggc cgactctggt
     4021 aacgacgctg acttatggtg ttcagtgctt tgctcgttat ccggaccata tgaagcagca
     4081 tgacttcttc aagtccgcca tgccggaagg ctatgtgcag gaacgcacga tttcctttaa
     4141 ggatgacggc acgtacaaaa cgcgtgcgga agtgaaattt gaaggcgata ccctggtaaa
     4201 ccgcattgag ctgaaaggca ttgactttaa agaggacggc aatatcctgg gccataagct
     4261 ggaatacaat tttaacagcc acaatgttta catcaccgcc gataaacaaa aaaatggcat
     4321 taaagcgaat tttaaaattc gccacaacgt ggaggatggc agcgtgcagc tggctgatca
     4381 ctaccagcaa aacactccaa tcggtgatgg tcctgttctg ctgccagaca atcactatct
     4441 gagcacgcaa agcgttctgt ctaaagatcc gaacgagaaa cgcgatcata tggttctgct
     4501 ggagttcgta accgcagcgg gcatcacgca tggtatggat gaactgtaca aatgaccagg
     4561 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     4621 tcggtgaacg ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt
     4681 tataggtctc aatcctaact cgaggccaat tggtgcggca attgataata acgaaaatgt
     4741 cttttaatga tctgggtata atgaggaatt ttccgaacgt ttttacttta tatatatata
     4801 tacatgtaac atatattcta tacgctatag agaaaggaaa tttttcaatt aaaaaaaaaa
     4861 tagagaaaga gtttcacttc ttgattatcg ctaacactaa tggttgaagt actgctactt
     4921 taattttatg ctgccaatga gacgacgggg tcatcacggc tcatcatgcg ccaaacaaat
     4981 gtgtgcaata cacgctcgga tgactgcatg atgaccgcac tgactgggga cagcagatcc
     5041 acctaagcct gtgagagaag cagacacccg acagatcaag gcagttaact agtgcactgc
     5101 agtacaaatc gtccccaaca aaagtgggct ctcaaaattc atcacattta aatgcatata
     5161 ggaagagcaa cagttggttt gcatctgatg ttccttaaag atttcgacat aatgtgcgaa
     5221 gtagatccct tcagcactgt acttgttttt gcgactagat tgtaaatcat tctttattta
     5281 atctctttct ttaactactg cttaaagtat aatttggtcc gtagtttaat aactatacta
     5341 agcgaacgct ctctcgaaat ttttcttaac gcgtccttgt actgcgtcta acgcttttgc
     5401 cacttggatt tctattatag gaaatagtct cacttactgg gcgacgaatt ttcgcgtttt
     5461 gatgaagcac aggaagaatt tctttatgct cagaagtttg acagcaagca agttcatcat
     5521 tcgaactagc cttattgttt tagttcagtg acagcgaact gccgtactcg atgctttatt
     5581 tctcacggta gagcggaaga acagatagat ccctacatat tgctaccact tctattacac
     5641 aatagtttca atagcttgca gcgtagctaa actctaaaat ttatctaaat cactcatata
     5701 aaccgaaccc ttccccttcc gcttatagta cagctgatgc tccttcacta ttttaacatg
     5761 tggaattctt gaaagaatga aatcgccatg ccaagccatc acacggtctt ttatgcaatt
     5821 gattgaccgc ctgcaacaca taggcagtaa aatttttaca agcagtagca gaacaggcca
     5881 cacaatcgca agtgattaac gtccacacag gtatagggtt tctggaccat atgatacatg
     5941 ctctggccaa gcattccggc tggtcgctaa tcgttgagtg ccgagcggcc gcgattatca
     6001 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6061 atatatgagt aaacttggtc tgacag
//
