LOCUS       sequence-380547-        6267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High 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 6267)
  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 6267)
  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: pRSII42H
FEATURES             Location/Qualifiers
     source          1..6267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      881..1078
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1166..1621)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1303..1324)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1515..1534
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(1598..1978)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWTRPVAAH"
     primer_bind     1747..1769
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1761..1778
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1762..1778
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1788..1807
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1788..1806
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1815..1922
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     1831..1847
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1832..1848
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1882..1898)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1882..1898)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1935..1955)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1935..1953)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1974..1990)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1974..1990)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1987..2009)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1998..2014
                     /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(2022..2028,2029..2046,2047..2052))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2067..2088
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2205..2222)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2376..2964)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2456..2475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3135..3926,3927..3995))
                     /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     3758..3777
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3996..4100)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4127..5473
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     5514..5532
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     5692..5711
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            join(5770..6267,1..1078)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        5770..6113
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             join(6114..6267,1..875)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             join(6114..6267,1..875)
                     /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
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
ORIGIN
        1 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
       61 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
      121 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
      181 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
      241 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
      301 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
      361 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
      421 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
      481 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
      541 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
      601 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
      661 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
      721 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
      781 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
      841 gacgccccag cactcgtccg agggcaaagg aataatcagt actgacaata aaaagattct
      901 tgttttcaag aacttgtcat ttgtatagtt tttttatatt gtagttgttc tattttaatc
      961 aaatgttagc gtgatttata ttttttttcg cctcgacatc atctgcccag atgcgaagtt
     1021 aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg ctatactgct
     1081 gtcgattcga tactaacgcc gccatccagt gtctatgcgg tgtgaaatac cgcacagatg
     1141 cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg
     1201 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     1261 tataaatcga aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     1321 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     1381 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     1441 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     1501 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     1561 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     1621 tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     1681 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     1741 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac
     1801 tatagggcga attgggtacc gggccccccc tcgaggtcga cggtatcgat aagcttgata
     1861 tcgaattcct gcagcccggg ggatccacta gttctagagc ggccgccacc gcggtggagc
     1921 tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     1981 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     2041 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     2101 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2161 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     2221 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2281 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcgaaag
     2341 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2401 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2461 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2521 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2581 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2641 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2701 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2761 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2821 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2881 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2941 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3001 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3061 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3121 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3181 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3241 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3301 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3361 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3421 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3481 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3541 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3601 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3661 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3721 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3781 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3841 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3901 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3961 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     4021 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4081 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaacg aagcatctgt
     4141 gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct
     4201 gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat
     4261 ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag
     4321 aatctgagct gcatttttac agaacagaaa tgcaacgcga gagcgctatt ttaccaacaa
     4381 agaatctata cttctttttt gttctacaaa aatgcatccc gagagcgcta tttttctaac
     4441 aaagcatctt agattacttt ttttctcctt tgtgcgctct ataatgcagt ctcttgataa
     4501 ctttttgcac tgtaggtccg ttaaggttag gagaaggcta ctttggtgtc tattttctct
     4561 tccataaaaa aagcctgact ccacttcccg cgtttactga ttactagcga agctgcgggt
     4621 gcattttttc aagataaagg catccccgat tatattctat accgatgtgg attgcgcata
     4681 ctttgtgaac agaaggtgat agcgttgatg attcttcatt ggtcagaaaa ttatgaacgg
     4741 tttcttctat tttgtctcta tatactacgt ataggaaatg tttacatttt cgtattgttt
     4801 tcgattcact ctatgaatag ttcttactac aatttttttg tctaaagagt aatactagag
     4861 ataaacataa aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg aaaggtggat
     4921 gggtaggtta tatagggata tagcacagag atatatagca aagagatact tttgagcaat
     4981 gtttgtggaa gcggtattcg caatatttta gtagctcgtt acagtccggt gcgtttttgg
     5041 ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg aagttcctat
     5101 actttctagc tagagaatag gaacttcgga ataggaactt caaagcgttt ccgaaaacga
     5161 gcgcttccga aaatgcaacg cgagctgcgc acatgcagct cactgttcac gtcgcaccta
     5221 tatctgcgtg ttgcctgtat atatatatac atgagaagaa cggcatagtg cgtgtttatg
     5281 cttaaatgcg tacttatatg cgtctattta tgtaggatga aaggtagtct agtacctcct
     5341 gtgatattat cccattccat gcggggtatc gtatgcttcc ttcagcacta ccctttagct
     5401 gttctatatg ctgccactcc tcaattggat tagtctcatc cttcaatgct atcatttcct
     5461 ttgatattgg atcatactaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5521 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacgca
     5581 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     5641 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     5701 agcagattgt actgagagtg caccatacca ccttagcttg cctcgtcccc gccgggtcac
     5761 ccggccagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca
     5821 ggggcatgat gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt
     5881 gcatccatac attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctgcgg
     5941 acctgcgagc agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc
     6001 ctgtagagaa atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct
     6061 tttaaaatct tgctaggata cagttctcac atcacatccg aacataaaca accatgggta
     6121 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     6181 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     6241 gagggcgtgg atatgtcctg cgggtaa
//
