LOCUS       sequence-380563-        6048 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High copy vector backbone with G418 selectivity, contains MCS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6048)
  AUTHORS   Smith JR, Sislak CD, Fernandez Mendoza P, Carmichael L, Lewis AG, 
            Chen A, Jiang GZ, Gibney PA
  TITLE     Auxotrophy-Independent Plasmid Shuttle Vectors for Applications in 
            Diverse Yeasts
  JOURNAL   Appl Microbiol. 2024; 4(1):453-469. doi: 
            10.3390/applmicrobiol4010031
REFERENCE   2  (bases 1 to 6048)
  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: "Appl 
            Microbiol."; date: "2024"; volume: " 4(1):453-469. doi"; pages: " 
            10.3390/applmicrobiol401003"
COMMENT     Sequence Label: pRSII42K
FEATURES             Location/Qualifiers
     source          1..6048
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      726..923
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1011..1466)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1148..1169)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1360..1379
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(1443..1823)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWTRPVAAH"
     primer_bind     1592..1614
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1606..1623
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1607..1623
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1633..1652
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1633..1651
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1660..1767
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     1676..1692
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1677..1693
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1780..1800)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1780..1798)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1819..1835)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1819..1835)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1832..1854)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1843..1859
                     /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(1867..1873,1874..1891,1892..1897))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1912..1933
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2050..2067)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2221..2809)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2301..2320)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2980..3771,3772..3840))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQRFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3603..3622
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3841..3945)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3972..5318
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     5359..5377
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5415..5437)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5537..5556
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            join(5615..6048,1..923)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        5615..5958
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             join(5959..6048,1..720)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPICNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGDFFPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(6026..6045)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg
       61 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
      121 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
      181 gatgcatggt tactcaccac tgcgatcccc ggcaaaacag cattccaggt attagaagaa
      241 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
      301 tcgattccta tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
      361 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
      421 tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc accggattca
      481 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
      541 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
      601 tggaactgcc tcggtgattt ttttccttca ttacagaaac ggctttttca aaaatatggt
      661 attgataatc ctgatatgaa taaattgcag tttcatttga tgcttgatga gtttttctaa
      721 tcagtactga caataaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt
      781 atattgtagt tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg
      841 acatcatctg cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta
      901 tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta acgccgccat ccagtgtcta
      961 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag
     1021 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     1081 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     1141 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     1201 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     1261 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     1321 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     1381 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     1441 gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa
     1501 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     1561 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     1621 agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag
     1681 gtcgacggta tcgataagct tgatatcgaa ttcctgcagc ccgggggatc caccagttct
     1741 agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc
     1801 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     1861 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     1921 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     1981 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2041 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2101 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2161 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2221 tttccatagg ctccgccccc ctgacgagca tcacaaaaat ggacgctcaa gtcagaggtg
     2281 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2341 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2401 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2461 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2521 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2581 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2641 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2701 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2761 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2821 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2881 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2941 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3001 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3061 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3121 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3181 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3241 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3301 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggct cccaacgatc
     3361 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3421 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3481 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3541 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3601 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3661 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3721 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3781 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaacgtt gaatactcat
     3841 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3901 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3961 agtgccacct gaacgaagca tctgtgcttc attttgtaga acaaaaatgc aacgcgagag
     4021 cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga
     4081 aagcgctatt ttaccaacga agaatctgtg cttcattttt gtaaaacaaa aatgcaacgc
     4141 gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa
     4201 cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct acaaaaatgc
     4261 atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc tcctttgtgc
     4321 gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag gttagaagaa
     4381 ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact tcccgcgttt
     4441 actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc ccgattatat
     4501 tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt tgatgattct
     4561 tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac tacgtatagg
     4621 aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt actacaattt
     4681 ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg agtttagatg
     4741 caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca cagagatata
     4801 tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaata ttttagtagc
     4861 tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc gcttttggtt
     4921 ttcaaaagcg ctctgaagtt cctatacttt ctagctagag aataggaact tcggaatagg
     4981 aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc tgcgcacata
     5041 cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata tatacgtgag
     5101 aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct atttatgtag
     5161 gatgaaaggt agtctagtac ctcctgtgat attatcccat tccatgcggg gtatcgtatg
     5221 cttccttcag cactaccctt tagctgttct atatgctgcc actcctcaat tggattagtc
     5281 tcatccttca atgctatcat ttcctttgat attggatcat actaagaaac cattattatc
     5341 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     5401 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     5461 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     5521 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca taccacctta
     5581 gcttgcctcg tccccgccgg gtcacccggc cagcgacatg gaggcccaga ataccctcct
     5641 tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc
     5701 catacatccc catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag
     5761 caaaaattac ggctcctcgc tgcagacctg cgagcaggga aacgctcccc tcacagacgc
     5821 gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact
     5881 gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac
     5941 atccgaacat aaacaaccat gggtaaggaa aagactcacg tttcgaggcc gcgattaaat
     6001 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataat
//
