LOCUS       sequence-234440-        7260 bp    DNA     circular SYN 15-APR-2025
DEFINITION  ADE2-NatMx ICE plasmid used to facilitate yeast recombination 
            cloning and stable genomic Integration after CEN Excision..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7260)
  AUTHORS   Flagg MP, Kao A, Hampton RY
  TITLE     ICE plasmids: combining the ease of yeast recombination cloning with
            the stability of genomic integration.
  JOURNAL   Yeast. 2019 May 10. doi: 10.1002/yea.3400.
  PUBMED    31074531
REFERENCE   2  (bases 1 to 7260)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Yeast. 2019
            May 10. doi: 10.1002/yea.3400."
COMMENT     Sequence Label: pICE ADE2-NatMx
FEATURES             Location/Qualifiers
     source          1..7260
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    11..514
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     complement(569..589)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(569..587)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(608..624)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(608..624)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(621..643)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    632..648
                     /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)."
     promoter        complement(join(656..662,663..680,681..686))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(839..856)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1010..1598)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1090..1109)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1769..2560,2561..2629))
                     /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     2392..2411
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2630..2734)
                     /gene="bla"
                     /label=AmpR promoter
     gene            2807..3929
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        2807..3150
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3151..3726
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVISYSAWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFAGERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGERQALYMSMPCP"
     terminator      3732..3929
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     3982..4000
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4038..4060)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4160..4179
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4208..4583
                     /gene="S. cerevisiae ADE2"
                     /label=ADE2 promoter
     CDS             4584..6299
                     /codon_start=1
                     /gene="S. cerevisiae ADE2"
                     /product="phosphoribosylaminoimidazole carboxylase, 
                     required for adenine biosynthesis"
                     /label=ADE2
                     /note="yeast auxotrophic marker"
                     /translation="MDSRTVGILGGGQLGRMIVEAANRLNIKTVILDAENSPAKQISNS
                     NDHVNGSFSNPLDIEKLAEKCDVLTIEIEHVDVPTLKNLQVKHPKLKIYPSPETIRLIQ
                     DKYIQKEHLIKNGIAVTQSVPVEQASETSLLNVGRDLGFPFVLKSRTLAYDGRGNFVVK
                     NKEMIPEALEVLKDRPLYAEKWAPFTKELAVMIVRSVNGLVFSYPIVETIHKDNICDLC
                     YAPARVPDSVQLKAKLLAENAIKSFPGCGIFGVEMFYLETGELLINEIAPRPHNSGHYT
                     IDACVTSQFEAHLRSILDLPMPKNFTSFSTITTNAIMLNVLGDKHTKDKELETCERALA
                     TPGSSVYLYGKESRPNRKVGHINIIASSMAECEQRLNYITGRTDIPIKISVAQKLDLEA
                     MVKPLVGIIMGSDSDLPVMSAACAVLKDFGVPFEVTIVSAHRTPHRMSAYAISASKRGI
                     KTIIAGAGGAAHLPGMVAAMTPLPVIGVPVKGSCLDGVDSLHSIVQMPRGVPVATVAIN
                     NSTNAALLAVRLLGAYDSSYTTKMEQFLLKQEEEVLVKAQKLETVGYEAYLENK"
     rep_origin      complement(6504..6959)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6641..6662)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6853..6872
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7089..7111
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7103..7120
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7104..7120
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7130..7149
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7130..7148
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     7173..7189
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     7174..7190
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7224..7240)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7224..7240)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 tcttaggacg gatcgcttgc ctgtaactta cacgcgcctc gtatctttta atgatggaat
       61 aatttgggaa tttactctgt gtttatttat ttttatgttt tgtatttgga ttttagaaag
      121 taaataaaga aggtagaaga gttacggaat gaagaaaaaa aaataaacaa aggtttaaaa
      181 aatttcaaca aaaagcgtac tttacatata tatttattag acaagaaaag cagattaaat
      241 agatatacat tcgattaacg ataagtaaaa tgtaaaatca caggattttc gtgtgtggtc
      301 ttctacacag acaagatgaa acaattcggc attaatacct gagagcagga agagcaagat
      361 aaaaggtagt atttgttggc gatcccccta gagtctttta catcttcgga aaacaaaaac
      421 tattttttct ttaatttctt tttttacttt ctatttttaa tttatatatt tatattaaaa
      481 aatttaaatt ataattattt ttatagcacg tgatgaaaag gacccaggtg gcgcggccgc
      541 caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
      601 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
      661 taggagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagg taactcacat
      721 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
      781 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
      841 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
      901 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
      961 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     1021 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     1081 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     1141 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1201 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1261 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1321 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1381 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1441 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1501 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1561 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1621 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     1681 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1741 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     1801 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     1861 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     1921 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     1981 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     2041 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     2101 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     2161 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2221 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     2281 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     2341 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     2401 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2461 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2521 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2581 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2641 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     2701 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     2761 acgtcttgtt tagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca
     2821 gaataccctc cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc
     2881 gtacatttag cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg
     2941 cacggcgcga agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc
     3001 cctcacagac gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta
     3061 ggatttgcca ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag
     3121 ttctcacatc acatccgaac ataaacaacc atgggtacca ctcttgacga cacggcttac
     3181 cggtaccgca ccagtgtccc gggggacgcc gaggccatcg aggcactgga tgggtccttc
     3241 accaccgaca ccgtcttccg cgtcaccgcc accggggacg gcttcaccct gcgggaggtg
     3301 ccggtggacc cgcccctgac caaggtgttc cccgacgacg aatcggacga cgaatcggac
     3361 gacggggagg acggcgaccc ggactcccgg acgttcgtcg cgtacgggga cgacggcgac
     3421 ctggcgggct tcgtggtcat ctcgtactcg gcgtggaacc gccggctgac cgtcgaggac
     3481 atcgaggtcg ccccggagca ccgggggcac ggggtcgggc gcgcgttgat ggggctcgcg
     3541 acggagttcg ccggcgagcg gggcgccggg cacctctggc tggaggtcac caacgtcaac
     3601 gcaccggcga tccacgcgta ccggcggatg gggttcaccc tctgcggcct ggacaccgcc
     3661 ctgtacgacg gcaccgcctc ggacggcgag cggcaggcgc tctacatgag catgccctgc
     3721 ccctaatcag tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt
     3781 ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc
     3841 gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca
     3901 atcgtatgtg aatgctggtc gctatactgc tgtcgattcg atactaacgc cgccatccag
     3961 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     4021 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4081 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4141 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatagatct
     4201 gaattaattc ttgaataata cataactttt cttaaaagaa tcaaagacag ataaaattta
     4261 agagatatta aatattagtg agaagccgag aattttgtaa caccaacata acactgacat
     4321 ctttaacaac ttttaattat gatacatttc ttacgtcatg attgattatt acagctatgc
     4381 tgacaaatga ctcttgttgc atggctacga accgggtaat actaagtgat tgactcttgc
     4441 tgacctttta ttaagaacta aatggacaat attatggagc atttcatgta taaattggtg
     4501 cgtaaaatcg ttggatctct cttctaagta catcctacta taacaatcaa gaaaaacaag
     4561 aaaatcggac aaaacaatca agtatggatt ctagaacagt tggtatatta ggagggggac
     4621 aattgggacg tatgattgtt gaggcagcaa acaggctcaa cattaagacg gtaatactag
     4681 atgctgaaaa ttctcctgcc aaacaaataa gcaactccaa tgaccacgtt aatggctcct
     4741 tttccaatcc tcttgatatc gaaaaactag ctgaaaaatg tgatgtgcta acgattgaga
     4801 ttgagcatgt tgatgttcct acactaaaga atcttcaagt aaaacatccc aaattaaaaa
     4861 tttacccttc tccagaaaca atcagattga tacaagacaa atatattcaa aaagagcatt
     4921 taatcaaaaa tggtatagca gttacccaaa gtgttcctgt ggaacaagcc agtgagacgt
     4981 ccctattgaa tgttggaaga gatttgggtt ttccattcgt cttgaagtcg aggactttgg
     5041 catacgatgg aagaggtaac ttcgttgtaa agaataagga aatgattccg gaagctttgg
     5101 aagtactgaa ggatcgtcct ttgtacgccg aaaaatgggc accatttact aaagaattag
     5161 cagtcatgat tgtgaggtct gttaacggtt tagtgttttc ttacccaatt gtagagacta
     5221 tccacaagga caatatttgt gacttatgtt atgcgcctgc tagagttccg gactccgttc
     5281 aacttaaggc gaagttgttg gcagaaaatg caatcaaatc ttttcccggt tgtggtatat
     5341 ttggtgtgga aatgttctat ttagaaacag gggaattgct tattaacgaa attgccccaa
     5401 ggcctcacaa ctctggacat tataccattg atgcttgcgt cacttctcaa tttgaagctc
     5461 atttgagatc aatattggat ttgccaatgc caaagaattt cacatctttc tccaccatta
     5521 caacgaacgc cattatgcta aatgttcttg gagacaaaca tacaaaagat aaagagctag
     5581 aaacttgcga aagagcattg gcgactccag gttcctcagt gtacttatat ggaaaagagt
     5641 ctagacctaa cagaaaagta ggtcacataa atattattgc ctccagtatg gcggaatgtg
     5701 aacaaaggct gaactacatt acaggtagaa ctgatattcc aatcaaaatc tctgtcgctc
     5761 aaaagttgga cttggaagca atggtcaaac cattggttgg aatcatcatg ggatcagact
     5821 ctgacttgcc ggtaatgtct gccgcatgtg cggttttaaa agattttggc gttccatttg
     5881 aagtgacaat agtctctgct catagaactc cacataggat gtcagcatat gctatttccg
     5941 caagcaagcg tggaattaaa acaattatcg ctggagctgg tggggctgct cacttgccag
     6001 gtatggtggc tgcaatgaca ccacttcctg tcatcggtgt gcccgtaaaa ggttcttgtc
     6061 tagatggagt agattcttta cattcaattg tgcaaatgcc tagaggtgtt ccagtagcta
     6121 ccgtcgctat taataatagt acgaacgctg cgctgttggc tgtcagactg cttggcgctt
     6181 atgattcaag ttatacaacg aaaatggaac agtttttatt aaagcaagaa gaagaagttc
     6241 ttgtcaaagc acaaaagtta gaaactgtcg gttacgaagc ttatctagaa aacaagtaat
     6301 atataagttt attgatatac ttgtacagca aataattata aaatgatata cctatttttt
     6361 aggctttgtt atgattacat caaatgtgga cttcatacat agaaatcaac gcttacaggt
     6421 gtcctttttt aagaatttca tacataagat ctatgcggtg tgaaataccg cacagatgcg
     6481 taaggagaaa ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt
     6541 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     6601 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     6661 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     6721 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     6781 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     6841 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     6901 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     6961 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     7021 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     7081 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc
     7141 actatagggc gaattgggta ccgggccccc cctcgaggtc gacggtatcg ataagcttga
     7201 tatcgaattc ctgcagcccg ggggatccac tagttctaga gcggccgccc tcttagcgct
//
