LOCUS       sequence-334691-        6811 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MATA fragment in pTOPO vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6811)
  AUTHORS   Xie ZX, Mitchell LA, Liu HM, Li BZ, Liu D, Agmon N, Wu Y, Li X, Zhou
            X, Li B, Xiao WH, Ding MZ, Wang Y, Yuan YJ, Boeke JD
  TITLE     Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of 
            Saccharomyces cerevisiae.
  JOURNAL   G3 (Bethesda). 2018 Jan 4;8(1):173-183. doi: 10.1534/g3.117.300347.
  PUBMED    29150593
REFERENCE   2  (bases 1 to 6811)
  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"; doi: "10.1534/g3.117"; 
            journalName: "G3 (Bethesda)"; date: "2018-01-4- 4"; volume: "8"; 
            issue: "1"; pages: "173-183"
COMMENT     Sequence Label: pXZX352
FEATURES             Location/Qualifiers
     source          1..6811
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..45
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    162..183
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(198..203,204..221,222..228)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    236..252
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     241..263
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     260..276
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     260..276
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        294..312
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     294..311
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3754..3773)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3755..3773)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3780..3797)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3780..3796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3789..3811)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3934..4236
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4115..4134
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4585..5379
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4585..5379
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4639..4658)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5249..5268
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      5579..5588
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5585..5959
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      6097..6685
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6586..6605
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
       61 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
      121 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
      181 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
      241 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctatttagg
      301 tgacactata gaatactcaa gctatgcatc aagcttggta ccgagctcgg atccactagt
      361 aacggccgcc agtgtgctgg aattcgccct tacgataact ggttggaaag cgtaaacacg
      421 gagtcaattg ttgtatcgtt gtgtttgaaa tgtcttgtga attctattgt ttcaatatcc
      481 aagtaaagag aagattctaa gagagtcaat gtctttgcaa gtgaccatgg agatacgttt
      541 agagcgaagg cttcgacttc tagaagttca ccctgtttcc attggaaaag taaatcactg
      601 aggtcagttg caccgcacaa ttcatcattt gcgttcgttt tgtctatctc gtattggaga
      661 aataagtttt cgtagtccag gggcggttta ttttcttcga tttccttatc agttagacca
      721 atgtaatgaa tcttcaattg cctcgttctt tttttgttta taaacagctg aactatgtct
      781 gcaaacagtt cttggtattc ataatattca gccaagtgac tgtaccaaaa attgaggata
      841 tcttttaatg ttgtctcact atcttgccaa taagactcta cccagatttg tattagacga
      901 gggacggagt gatttttgtg tttgttttta ttaattgtgg gataggatag tagcaactct
      961 tggaggagag cattgtcagt tgtccagtct ctgaagttaa gtagtaagtt tgcggagtca
     1021 aagggggatg gcttttgcca tttgtgagag ttgtgcggca gcatcttatt caaatagagc
     1081 tgtattctga agacctcttg tagaacatca tccatactaa aaagtaaatc gtcctgtccc
     1141 attacgagct gtattagtgc tgtgaccctc tgtatattta cgttgccatg aagaaggtaa
     1201 tgggcgatat tttgatacaa ttcctgagtt gcatgttgga ttgagtttac gaagggtcgc
     1261 cagacggcca gaaacctcca ggcggagtta acaactaata atacggcatc catgtttgca
     1321 tcagcgccga gcctatacca gtcactgagt agacgttttc ttgctctttt tatgtcctga
     1381 cttcttttga cgagggggca ttctctagag acacaggcag ttgcttccag caactgccgt
     1441 acggccgttc tcatgctgtc gaggattttt tttgggacga tattgtcatt atagggcagt
     1501 gtgtgactta tgaattgttg tagaaggacg tctgtgatgt tggagatatg tattttgtta
     1561 actcttcttg agacgatttg gccctggata gcgaagcgtg cggttacaaa taggtcgtct
     1621 tgttcaagaa ggtaggcgag gacattatct atcagtacaa acatcttagt agtgtctgag
     1681 gagagggttg attgtttatg tatttttgcg aaatatatat atatatattc tacacagata
     1741 tatacatatt tgtttttcgg gctcattctt tcttctttgc cagaggctca ccgctcaaga
     1801 ggtccgctaa ttctggagcg attgttattg ttttttcttt tcttcttcta ttcgaaaccc
     1861 agtttttgat ttgaatgcga gataaactgg tattcttcat tagattctct aggcccttgg
     1921 tatctagata tgggttctcg atgttctttg caaaccaact ttctagtatt cggacatttt
     1981 cttttgtaaa ccggtgtcct ctgtaaggtt tagtactttt gtttatcata tcttgagtta
     2041 ccacattaaa taccaaccca tccgccgatt tatttttctg tgtaagttga taattacttc
     2101 tatcgttttc tatgctgcgc atttctttga gtaatacagt aatggtagta gtgagttgag
     2161 atgttgtttg caacaacttc ttctcctcat cactaatctt acggtttttg ttggccctag
     2221 ataagaatcc taatatatcc cttaattcaa cttcttcttc tgttgttaca ctctctggta
     2281 acttaggtaa attacagcaa atagaaaaga gctttttatt cttgattttt gttctttcgg
     2341 ggaaactgta taaaacttcc aaaaaggaaa agtaaaacaa tacatctcct tatatcaaag
     2401 aaaatcaaga aggacaacat ggatgatatt tgtagtatgg cggaaaacat aaacagaact
     2461 ctgtttaaca ttctaggtac tgagattgat gaaatcaatc tcaatactaa taatctttat
     2521 aatgtatgtt ttcatttcaa ggatagcctt tgaatcaatt tactaacaat acttcagttt
     2581 ataatggaaa gtaatttgac taaagtagag caacatacat tacacaaaaa tatttctaac
     2641 aataggttag aaatatacca ccacattaaa aaagagaaga gcccaaaggg aaaatcatca
     2701 atatcacccc aagcacgggc atttttagaa caggttttta gaagaaagca aagccttaat
     2761 tccaaggaaa aagaagaagt tgcaaagaaa tgtggcatta ctccacttca agtaagagtt
     2821 tgggtatgta atatgagaat caaacttaaa tatatcctat actaacaatt tgtagttcat
     2881 aaataaacgt atgagatcta aataaattcg ttttcaatga ttaaaatagc atagtcgggt
     2941 ttttctttta gtttcagctt tccgcaacag taaaatttta taaaccctgg ttttggtttt
     3001 gtagagtggt tgacgaataa ttatgctgaa gtacgtggtg acggatattg ggaagatgtg
     3061 tttgtacatt tggccttata gagtgtggtc gtggcggagg ttgtttatct ttcgagtact
     3121 gaatgttgtc agtatagcta tcctatttga aactccccat cgtcttgctc ttgttcccaa
     3181 tgtttgttta tacactcata tggctatacc cttatctact tgcctctttt gtttatgtct
     3241 atgtatttgt ataaaatatg atattactca gactcaagca aacaatcaat tcttagcatc
     3301 attctttgtt cttatcttaa ccataaacga tcttgatgtg acttttgtaa tttgaacgaa
     3361 ttggctatac gggacggatg acaaatgcac cattactcta ggttgttgtt ggatcttaac
     3421 aaaccgtaaa ggtaaactgc ccatgcggtt cacatgactt ttgactttcc tttgtttgct
     3481 agttaccttc ggcttcacaa tttgtttttc cacttttcta acaggtttat cacctttcaa
     3541 acttatcttt atcttattcg ccttcttggg tgcctccaca gtagaggtta cttccttttt
     3601 aatatgtact tttaggatac tttcacgctt tataacaata tcaagtttac cttcttcatt
     3661 actattcatc ttcgccacaa gtctaagggc gaattctgca gatatccatc acactggcgg
     3721 ccgctcgagc atgcatctag agggcccaat tcgccctata gtgagtcgta ttacaattca
     3781 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     3841 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     3901 ccttcccaac agttgcgcag cctatacgta cggcagttta aggtttacac ctataaaaga
     3961 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gccggggcga
     4021 cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt
     4081 tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt
     4141 gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
     4201 aaaaacgcca ttaacctgat gttctgggga atataaatgt caggcatgag attatcaaaa
     4261 aggatcttca cctagatcct tttcacgtag aaagccagtc cgcagaaacg gtgctgaccc
     4321 cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc aaagagaaag
     4381 caggtagctt gcagtgggct tacatggcga tagctagact gggcggtttt atggacagca
     4441 agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta
     4501 aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa
     4561 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4621 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4681 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4741 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     4801 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4861 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4921 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4981 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     5041 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     5101 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     5161 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     5221 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     5281 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     5341 cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc
     5401 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat caggtggcac
     5461 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5521 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg tgaggagggc
     5581 caccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc
     5641 ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc
     5701 cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc
     5761 ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc
     5821 ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga
     5881 gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt
     5941 ggccgaggag caggactgac acgtgctaaa acttcatttt taatttaaaa ggatctaggt
     6001 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6061 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6121 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6181 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6241 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6301 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6361 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6421 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6481 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6541 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6601 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6661 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6721 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     6781 ccgtattacc gcctttgagt gagctgatac c
//
