LOCUS       sequence-380566-        5434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low copy vector backbone with hygromycin B selectivity, contains 
            MCS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5434)
  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 5434)
  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: pRSII41H
FEATURES             Location/Qualifiers
     source          1..5434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      880..1077
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1192..1620)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1302..1323)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1514..1533
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(1597..1977)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWTHPVAAH"
     primer_bind     1746..1768
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1760..1777
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1761..1777
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1787..1806
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1787..1805
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1814..1921
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     1830..1846
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1831..1847
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1881..1897)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1881..1897)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1934..1954)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1934..1952)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1973..1989)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1973..1989)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1986..2008)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1997..2013
                     /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(2021..2027,2028..2045,2046..2051))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2066..2087
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2204..2221)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2375..2963)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2455..2474)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3134..3925,3926..3994))
                     /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     3757..3776
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3995..4099)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    4136..4639
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     4680..4698
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4736..4758)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4858..4877
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            join(4936..5434,1..1077)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        4936..5279
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             join(5280..5434,1..874)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSEAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
ORIGIN
        1 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
       61 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat
      121 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
      181 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
      241 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacgtggc gtgatttcat
      301 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
      361 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
      421 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
      481 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
      541 catcttcttc tggagaccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
      601 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
      661 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
      721 tcgatgcgac gcaatcgtcc gatccgaagc cgggactgtc gggcgtacac aaatcgcccg
      781 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
      841 acgccccagc actcgtccga gggcaaagga ataatcagta ctgacaataa aaagattctt
      901 gttttcaaga acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca
      961 aatgttagcg tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta
     1021 agtgcgcaga aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg
     1081 tcgattcgat actaacgccg ccatccagtg tctatgcggt gtgaaatacc gcacagatgc
     1141 gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta aaattcgcgt
     1201 taaacttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     1261 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     1321 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     1381 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     1441 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     1501 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     1561 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggtgcgt
     1621 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     1681 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     1741 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     1801 atagggcgaa ttgggtaccg ggccccccct cgaggtcgac ggtatcgata agcttgatat
     1861 cgaattcctg cagcccgggg gatccactag ttctagagcg gccgccaccg cggtggagct
     1921 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     1981 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     2041 taaagtgtaa agcctgaggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     2101 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2161 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2221 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2281 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2341 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2401 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2461 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2521 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2581 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2641 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2701 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2761 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     2821 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2881 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2941 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3001 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3061 cctagatcct tttaaattaa aaatgatgtt ttaaatcaat ctaaagtata tatgagtaaa
     3121 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3181 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3241 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3301 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3361 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3421 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3481 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3541 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3601 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3661 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3721 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3781 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3841 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3901 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     3961 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4021 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4081 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgggtcc ttttcatcac
     4141 gtgctataaa aataattata atttaaattt tttaatataa atatataaat taaaaataga
     4201 aagtaaaaaa ggaaattaaa gaaaaaatag tttttgtttt ccgaagatgt aaaagactct
     4261 agggggatcg ccaacaaata ctacctttta tcttgctctt cctgctctca ggtattaatg
     4321 ccgaattgtt tcatcttgtc tgtgtagaag accacacacg aaaatcctgt gattttacat
     4381 tttacttatc gttaatcgaa tgtatatcta tttaatctgc ttttcttgtc taataaatat
     4441 atatgtaaag tacgcttttt gttgaaattt tttaaacctt tgtttatttt tttttcttca
     4501 ttccgtaact cttctacctt ctttatttac tttctaaaat ccaaatacaa aacataaaaa
     4561 taaataaaca cagagtaaat tcccaaatta ttccatcatt aaaagatacg aggcgcgtgt
     4621 aagttacagg caagcgatcc gtctaagaaa ccattattat catgacatta acctataaaa
     4681 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     4741 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     4801 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     4861 catcagagca gattgtactg agagtgcacc ataccacctt agcttgcctc gtccccgccg
     4921 ggtcacccgg ccagcgacat ggaggcccag aataccctcc ttgacagtct tgacgtgcgc
     4981 agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc ccatgtataa
     5041 tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg
     5101 ctgcggacct gcgagcaggg aaacgctccc ctcacagacg cgttgaattg tccccacgcc
     5161 gcgcccctgt agagaaatat aaaaggttag gatttgccac tgaggttctt ctttcatata
     5221 cttcctttta aaatcttgct aggatacagt tctcacatca catccgaaca taaacaacca
     5281 tgggtaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     5341 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     5401 atgtaggagg gcgtggatat gtcctgcggg taaa
//
