LOCUS       sequence-477887-        6882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Selection plasmid for Rubisco directed evolution.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6882)
  AUTHORS   McDonald JL, Shapiro NP, Mengiste AA, Kaines S, Whitney SM, Wilson 
            RH, Shoulders MD
  TITLE     In vivo directed evolution of an ultrafast Rubisco from a 
            semianaerobic environment imparts oxygen resistance.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Jul 8;122(27):e2505083122. doi: 
            10.1073/pnas.2505083122. Epub 2025 Jun 30.
  PUBMED    40587785
REFERENCE   2  (bases 1 to 6882)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2025-07-8"; volume: "122(27):e2505083122. 
            doi"; pages: " 10.1073/pnas.2505083122. Epub 2025 Jun 30"
COMMENT     Sequence Label: pACYC-prk:nptII
FEATURES             Location/Qualifiers
     source          1..6882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..546
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     447..466
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     663..680
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1072..1174
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1175..1834
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1241..1260)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(3670..4548)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        4575..4859
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     4782..4801
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             4886..4908
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5916..6707
                     /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="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5967..5986)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6577..6596
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca
       61 gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta actggcttgg
      121 aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc atgacttcaa
      181 gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt
      241 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac tgaacggggg
      301 gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg
      361 gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa ggcaggaaca
      421 ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag tcctgtcggg
      481 tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta
      541 tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt
      601 ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc
      661 gtagcgagtc agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca
      721 ccggtgcagc cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc
      781 aacatagtaa gccagtatac actccgctag cgctgatgtc cggcggtgct tttgccgtta
      841 cgcaccaccc cgtcagtagc tgaacaggag ggacagctga tagaaacaga agccactgga
      901 gcacctcaaa aacaccatca tacactaaat cagtaagttg gcagcatcac ccgacgcact
      961 ttgcgccgaa taaatacctg tgacggaaga tcacttcgca gaataaataa atcctggtgt
     1021 ccctgttgat accgggaagc cctgggccaa cttttggcga aaatgagacg ttgatcggca
     1081 cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg tattttttga
     1141 gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
     1201 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
     1261 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
     1321 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     1381 tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
     1441 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     1501 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     1561 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     1621 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     1681 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     1741 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca
     1801 acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc
     1861 ttaaacgcct ggttgctacg cctgaataag tgataataag cggatgaatg gcagaaattc
     1921 gaaagcaaat tcgacccggt cgtcggttca gggcagggtc gttaaatagc cgcttatgtc
     1981 tattgctggt ttaccggttt attgactacc ggaagcagtg tgaccgtgtg cttctcaaat
     2041 gcctgaggcc agtttgctca ggctctcccc gtggaggtaa taattgacga tatgatcatt
     2101 tattctgcct cccagagcct gataaaaacg gttagcgctt cgttaataca gatgtaggtg
     2161 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     2221 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgctgccg
     2281 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     2341 tgccccgcgc ccaccggaag gagctaccgg cagcggtgcg gactgttgta actcagaata
     2401 agaaatgagg ccgctcatgg cgttgactct cagtcatagt atcgtggtat caccggttgg
     2461 ttccactctc tgttgcgggc aacttcagca gcacgtaggg gacttccgcg tttccagact
     2521 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     2581 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     2641 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca
     2701 ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga
     2761 tatgttctgc caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc
     2821 aattcttgga gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg
     2881 gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct
     2941 acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc
     3001 agcggtccaa tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc
     3061 tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg
     3121 ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc
     3181 aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     3241 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     3301 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     3361 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     3421 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     3481 ggcatcggtc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     3541 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcataatgtg cctgtcaaat
     3601 ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt
     3661 cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg
     3721 gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc
     3781 gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag
     3841 cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg
     3901 gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat
     3961 atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt
     4021 atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc
     4081 aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat
     4141 ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt
     4201 attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta
     4261 aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc
     4321 tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac
     4381 caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc
     4441 gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc
     4501 attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac ttttcatact
     4561 cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc gtcactgcgt
     4621 cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa gcattctgta
     4681 acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata atcacggcag
     4741 aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag catttttatc
     4801 cataagatta gcggatccta cctgacgctt tttatcgcaa ctctctactg tttctccata
     4861 cccgtttttt tgggctagaa ataattttgt ttaactttaa gaaggagata tacataccca
     4921 tggctaaacc tgatcgtgta gttcttatcg gtgttgctgg tgattcaggt tgtggaaaaa
     4981 gcacttttct gaatcgtttg gctgatcttt tcgggaccga attaatgact gtaatttgcc
     5041 tagatgacta tcatagttta gatagaaaag gccgtaaaga ggcaggtgta acagcccttg
     5101 atccacgagc aaataacttc gatttgatgt acgaacaagt taaggcgctg aaaaacggag
     5161 aaacgattat gaaaccgata tacaaccacg agactggttt gatcgatccc ccagaaaaaa
     5221 tcgagcctaa tcggattatt gttatagagg gattacatcc tttgtatgac gaaagagtac
     5281 gtgagctgct agatttttcc gtatatcttg atattgatga tgaagtaaaa atcgcatgga
     5341 aaatccagag agacatggcc gaacgtggtc attcctatga agatgtgttg gcgtctatcg
     5401 aagcacgacg ccctgacttt aaagcatata tcgaacctca aagaggtcat gctgatattg
     5461 tcattcgtgt tatgccaact caactaattc cgaatgatac cgaacgtaaa gttctacgag
     5521 ttcagctgat tcaaagagaa ggacgtgatg gtttcgagcc cgcttacctg tttgatgaag
     5581 gtagcacaat tcaatggaca ccttgtggtc gtaaacttac ttgctcttat cctggaatcc
     5641 gtttggcgta tgggcctgat acctactatg gacatgaagt gtcagtacta gaggttgatg
     5701 ggcagtttga aaatttagaa gaaatgattt acgtagaagg gcacttaagt aaaacagata
     5761 cacaatacta tggtgaattg acccaccttt tattgcaaca taaagattat ccaggttcta
     5821 acaatggtac tggtttattc caggtattga ctggtcttaa gatgcgtgca gcttatgaac
     5881 gccttacttc tcaagccgct cctgttgcag ctagcattga acaagatgga ttgcacgcag
     5941 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     6001 gctgctctga tgccgccgtg tttcggctgt cagcgcaggg gcgcccggtt ctttttgtca
     6061 agaccgacct gtccggtgcc ctgaatgaac tccaggacga ggcagcgcgg ctatcgtggc
     6121 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     6181 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     6241 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     6301 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     6361 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     6421 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acacatggcg
     6481 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     6541 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     6601 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     6661 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggtctagggg
     6721 cccgtcgact gcagaggcct gcatgcaagc ttatcgatga taagctgtca aacatgagaa
     6781 ttacaactta tatcgtatgg ggctgacttc aggtgctaca tttgaagaga taaattgcac
     6841 tgaaatctag aaatatttta tctgattaat aagatgatct tc
//
