LOCUS       sequence-334683-        6916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MAT-alpha (alpha) fragment in pTOPO vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6916)
  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 6916)
  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: pXZX353
FEATURES             Location/Qualifiers
     source          1..6916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    520..541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(556..561,562..579,580..586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    594..610
                     /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     599..621
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     618..634
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     618..634
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        652..670
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     652..669
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4217..4236)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4218..4236)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4243..4260)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4243..4259)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4252..4274)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             4397..4699
                     /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     4578..4597
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             5048..5842
                     /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             5048..5842
                     /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(5102..5121)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5712..5731
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      6042..6051
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6048..6422
                     /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      join(6560..6916,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      541 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      601 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc tatttaggtg
      661 acactataga atactcaagc tatgcatcaa gcttggtacc gagctcggat ccactagtaa
      721 cggccgccag tgtgctggaa ttcgccctta cgataactgg ttggaaagcg taaacacgga
      781 gtcaattgtt gtatcgttgt gtttgaaatg tcttgtgaat tctattgttt caatatccaa
      841 gtaaagagaa gattctaaga gagtcaatgt ctttgcaagt gaccatggag atacgtttag
      901 agcgaaggct tcgacttcta gaagttcacc ctgtttccat tggaaaagta aatcactgag
      961 gtcagttgca ccgcacaatt catcatttgc gttcgttttg tctatctcgt attggagaaa
     1021 taagttttcg tagtccaggg gcggtttatt ttcttcgatt tccttatcag ttagaccaat
     1081 gtaatgaatc ttcaattgcc tcgttctttt tttgtttata aacagctgaa ctatgtctgc
     1141 aaacagttct tggtattcat aatattcagc caagtgactg taccaaaaat tgaggatatc
     1201 ttttaatgtt gtctcactat cttgccaata agactctacc cagatttgta ttagacgagg
     1261 gacggagtga tttttgtgtt tgtttttatt aattgtggga taggatagta gcaactcttg
     1321 gaggagagca ttgtcagttg tccagtctct gaagttaagt agtaagtttg cggagtcaaa
     1381 gggggatggc ttttgccatt tgtgagagtt gtgcggcagc atcttattca aatagagctg
     1441 tattctgaag acctcttgta gaacatcatc catactaaaa agtaaatcgt cctgtcccat
     1501 tacgagctgt attagtgctg tgaccctctg tatatttacg ttgccatgaa gaaggtaatg
     1561 ggcgatattt tgatacaatt cctgagttgc atgttggatt gagtttacga agggtcgcca
     1621 gacggccaga aacctccagg cggagttaac aactaataat acggcatcca tgtttgcatc
     1681 agcgccgagc ctataccagt cactgagtag acgttttctt gctcttttta tgtcctgact
     1741 tcttttgacg agggggcatt ctctagagac acaggcagtt gcttccagca actgccgtac
     1801 ggccgttctc atgctgtcga ggattttttt tgggacgata ttgtcattat agggcagtgt
     1861 gtgacttatg aattgttgta gaaggacgtc tgtgatgttg gagatatgta ttttgttaac
     1921 tcttcttgag acgatttggc cctggatagc gaagcgtgcg gttacaaata ggtcgtcttg
     1981 ttcaagaagg taggcgagga cattatctat cagtacaaac atcttagtag tgtctgagga
     2041 gagggttgat tgtttatgta tttttgcgaa atatatatat atatattcta cacagatata
     2101 tacatatttg tttttcgggc tcattctttc ttctttgcca gaggctcacc gctcaagagg
     2161 tccgctaatt ctggagcgat tgttattgtt ttttcttttc ttcttctatt cgaaacccag
     2221 tttttgattt gaatgcgaga taaactggta ttcttcatta gattctctag gcccttggta
     2281 tctagatatg ggttctcgat gttctttgca aaccaacttt ctagtattcg gacattttct
     2341 tttgtaaacc ggtgtcctct gtaaggttta gtacttttgt ttatcatatc ttgagttacc
     2401 acattaaata ccaacccatc cgccgattta tttttctgtg taagttgata attacttcta
     2461 tcgttttcta tgctgcgcat ttctttgagt aatacagtaa tggtagtagt gagttgagat
     2521 gttgtttgca acaacttctt ctcctcatca ctaatcttac ggtttttgtt ggccctagat
     2581 aagaatccta atatatccct taattcaact tcttcttctg ttgttacact ctctggtaac
     2641 ttaggtaaat tacagcaaat agaaaagagc tttttattta tgtctagtat gctggattta
     2701 aactcatctg tgatttgtgg atttaaaagg tctttaatgg gtattttatt cattttttct
     2761 tgcttatctt cctttttttc ttgcccactt ctaagctgat ttcaatctct cctttatata
     2821 tatttttaag ttccaacatt ttatgtttca aaacattaat gatgtctggg ttttgtttgg
     2881 gatgcaattt attgcttccc aatgtagaaa agtacatcat atgaaacaac ttaaactctt
     2941 aactacttct tttaaccttc actttttatg aaatgtatca accatatata ataacttaat
     3001 agacgacatt cacaatatgt ttacttcgaa gcctgctttc aaaattaaga acaaagcatc
     3061 caaatcatac agaaacacag cggtttcaaa aaagctgaaa gaaaaacgtc tagctgagca
     3121 tgtgaggcca agctgcttca atattattcg accactcaag aaagatatcc agattcctgt
     3181 tccttcctct cgatttttaa ataaaatcca aattcacagg atagcgtctg gaagtcaaaa
     3241 tactcagttt cgacagttca ataagacatc tataaaatct tcaaagaaat atttaaactc
     3301 atttatggct tttagagcat attactcaca gtttggctcc ggtgtaaaac aaaatgtctt
     3361 gtcttctctg ctcgctgaag aatggcacgc ggacaaaatg cagcacggaa tatgggacta
     3421 cttcgcgcaa cagtataatt ttataaaccc tggttttggt tttgtagagt ggttgacgaa
     3481 taattatgct gaagtacgtg gtgacggata ttgggaagat gtgtttgtac atttggcctt
     3541 atagagtgtg gtcgtggcgg aggttgttta tctttcgagt actgaatgtt gtcagtatag
     3601 ctatcctatt tgaaactccc catcgtcttg ctcttgttcc caatgtttgt ttatacactc
     3661 atatggctat acccttatct acttgcctct tttgtttatg tctatgtatt tgtataaaat
     3721 atgatattac tcagactcaa gcaaacaatc aattcttagc atcattcttt gttcttatct
     3781 taaccataaa cgatcttgat gtgacttttg taatttgaac gaattggcta tacgggacgg
     3841 atgacaaatg caccattact ctaggttgtt gttggatctt aacaaaccgt aaaggtaaac
     3901 tgcccatgcg gttcacatga cttttgactt tcctttgttt gctagttacc ttcggcttca
     3961 caatttgttt ttccactttt ctaacaggtt tatcaccttt caaacttatc tttatcttat
     4021 tcgccttctt gggtgcctcc acagtagagg ttacttcctt tttaatatgt acttttagga
     4081 tactttcacg ctttataaca atatcaagtt taccttcttc attactattc atcttcgcca
     4141 caagtctaag ggcgaattct gcagatatcc atcacactgg cggccgctcg agcatgcatc
     4201 tagagggccc aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca
     4261 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     4321 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     4381 cagcctatac gtacggcagt ttaaggttta cacctataaa agagagagcc gttatcgtct
     4441 gtttgtggat gtacagagtg atattattga cacgccgggg cgacggatgg tgatccccct
     4501 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat
     4561 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat
     4621 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct
     4681 gatgttctgg ggaatataaa tgtcaggcat gagattatca aaaaggatct tcacctagat
     4741 ccttttcacg tagaaagcca gtccgcagaa acggtgctga ccccggatga atgtcagcta
     4801 ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg
     4861 gcttacatgg cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc
     4921 agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt
     4981 gccgccaagg atctgatggc gcaggggatc aagctctgat caagagacag gatgaggatc
     5041 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     5101 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     5161 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     5221 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     5281 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     5341 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     5401 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     5461 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     5521 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     5581 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     5641 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     5701 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     5761 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     5821 ccttcttgac gagttcttct gaattattaa cgcttacaat ttcctgatgc ggtattttct
     5881 ccttacgcat ctgtgcggta tttcacaccg catcaggtgg cacttttcgg ggaaatgtgc
     5941 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6001 aataaccctg ataaatgctt caataatagc acgtgaggag ggccaccatg gccaagttga
     6061 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
     6121 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
     6181 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
     6241 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
     6301 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
     6361 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
     6421 gacacgtgct aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6481 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6541 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6601 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6661 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6721 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6781 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6841 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6901 ccagcttgga gcgaac
//
