LOCUS       sequence-487709-        9036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single copy expression vector for GWS1B Rubisco.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9036)
  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 9036)
  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: BAC-GWS1B
FEATURES             Location/Qualifiers
     source          1..9036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(186..290)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     399..418
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        399..417
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(424..429,430..445,446..452)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             1868..1885
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        join(2198..2203,2204..2220,2221..2226)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             2272..3474
                     /codon_start=1
                     /gene="tetA from transposon Tn10"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="NSSTKIALVITLLDAMGIGLIMPVLPTLLREFIASEDIANHFGVL
                     LALYALMQVIFAPWLGKMSDRFGRRPVLLLSLIGASLDYLLLAFSSALWMLYLGRLLSG
                     ITGATGAVAASVIADTTSASQRVKWFGWLGASFGLGLIAGPIIGGFAGEISPHSPFFIA
                     ALLNIVTFLVVMFWFRETKNTRDNTDTEVGVETQSNSVYITLFKTMPILLIIYFSAQLI
                     GQIPATVWVLFTENRFGWNSMMVGFSLAGLGLLHSVFQAFVAGRIATKWGEKTAVLLGF
                     IADSSAFAFLAFISEGWLVFPVLILLAGGGIALPALQGVMSIQTKSHQQGALQGLLVSL
                     TNATGVIGPLLFAVIYNHSLPIWDGWIWIIGLAFYCIIILLSMTFMLTPQAQGSKQETS
                     A"
     primer_bind     complement(3500..3518)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    3799..4272
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
     CDS             complement(4345..5316)
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFADKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     CDS             complement(5316..6491)
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGPIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     misc_feature    6817..7067
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(7070..7825)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEIGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      7916..8135
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             complement(join(8361..9036,1..116,117..185))
                     /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     8984..9003
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
       61 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
      121 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
      181 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
      241 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
      301 cgaaaagtgc cacctggcgg ccgcctttca gcaaaaaacc ccgcgagacc cccgaagagg
      361 ccccgcgggg ttatgctagg tcgacggagc tcgaattcta atacgactca ctatagggag
      421 accttgacaa ttaatcatcg gctcgtataa tgccagaagc ttagcaaaag ctaaaaccag
      481 gagctattta atggaccaaa gtaatcgtta cgccgactta tccttgaaag aggaggactt
      541 gatcaagggg gggaatcata tcctggttgc ctatacaatg gaacccgctg ccggggtcgg
      601 atatcttgaa gcagccgccc acattgccgc agagagttct actggaacca atgtggaggt
      661 ctcgactacg gatgaattta caaaaggggt cgatgctctt gtgtatttca ttgatgaggc
      721 caagggcatc atgaaggtcg cgtatcctaa tgatctgttc gatcgcaatg ttaccgatgg
      781 gcgcgtgatg atcgtttcct tcttgaccct ttgtattgga aataatcaag gaatgggaga
      841 cattgcaaat ctgcagatgc aggacttcta cgttcccccg cgtatgttgc aactgtttga
      901 cgggcctgcg aaggatattt cagatttatg gcgcatcttg ggtcgtccag ttaaagatgg
      961 tgggtacatc tcaggaacaa ttattaaacc gaaattaggt cttcgtcccg aacctttcgc
     1021 taaggcagcc tatcaatttt ggcttggcgg tgattttatc aagaatgatg agccccaagg
     1081 taaccaggta ttttgcccaa tcaagaaagt cctgcccttg gtttacgatt cgttaaagcg
     1141 cgcccaggac gaaactgggc aagccaaatt gtttagtatg aacatcacgg cagatgatca
     1201 ttacgagatg tgcgcgcgcg ctgactttgc ccttgaaacc ttcgggccgg atgctgacaa
     1261 agtggccttt cttgttgacg gttttgtcgg tgggccgggg atggttacaa cagcccgtcg
     1321 ccaatatccc tcccaatact tgcactacca ccgtgcaggt cacggcatgg tgactagccc
     1381 atcctctaag cgcggataca cggcctttgt gctggcgaag atgagtcgtt tacaaggggc
     1441 atccgggatc catgttggca ctatgggata cggaaaaatg gaaggcggca aagacgatcg
     1501 cattattgcc tacatgatcg agcgtgatag ctgcacggga ccattctatc atcaagagtg
     1561 gtatggtatg aagcctacaa ctccaattat ttctggaggt atgaacgctt tgcgtttgcc
     1621 tggcttcttc gagaatctgg gacacggaaa cgtaattaac actgctggcg gtggaagcta
     1681 cggccatatc gactcgccag cggcaggagc tgtctcgtta cgccaggcct atgaatgttg
     1741 gaaagctggg gccgatccta tcgagtgggc taaggagcac aaggaattcg cgcgtgcgtt
     1801 tgaatccttt ccaaaagacg cggacaaatt gttccccgga tggcgcgaaa aattaggagt
     1861 acataaacac caccaccacc accactagtg gtttaattaa cgcagcctga atggcgaata
     1921 gaagtttaaa cgctagacct agcataaccc cgcggggcct cttcgggggt ctcgcggggt
     1981 tttttgctga aaggctagac ctagcataac cccgcggggc ctcttcgggg gtctcgcggg
     2041 gttttttgct gaaaggctag acctagcata accccgcggg gcctcttcgg gggtctcgcg
     2101 gggttttttg ctgaaaggct agacctagca taaccccgcg gggcctcttc gggggtctcg
     2161 cggggttttt tgctgaaagg ctaggatata ttgatccttg acagcttatc atcgataagc
     2221 tttaatgcgg tagtttatca cagttgctaa cgcagtcagg caccgtgtac gaatagttcg
     2281 acaaagatcg cattggtaat tacgttactc gatgccatgg ggattggcct tatcatgcca
     2341 gtcttgccaa cgttattacg tgaatttatt gcttcggaag atatcgctaa ccactttggc
     2401 gtattgcttg cactttatgc gttaatgcag gttatctttg ctccttggct tggaaaaatg
     2461 tctgaccgat ttggtcggcg cccagtgctg ttgttgtcat taataggcgc atcgctggat
     2521 tacttattgc tggctttttc aagtgcgctt tggatgctgt atttaggccg tttgctttca
     2581 gggatcacag gagctactgg ggctgtcgcg gcatcggtca ttgccgatac cacctcagct
     2641 tctcaacgcg tgaagtggtt cggttggtta ggggcaagtt ttgggcttgg tttaatagcg
     2701 gggcctatta ttggtggttt tgcaggagag atttcaccgc atagtccctt ttttatcgct
     2761 gcgttgctaa atattgtcac tttccttgtg gttatgtttt ggttccgtga aaccaaaaat
     2821 acacgtgata atacagatac cgaagtaggg gttgagacgc aatcgaattc ggtatacatc
     2881 actttattta aaacgatgcc cattttgttg attatttatt tttcagcgca attgataggc
     2941 caaattcccg caacggtgtg ggtgctattt accgaaaatc gttttggatg gaatagcatg
     3001 atggttggct tttcattagc gggtcttggt cttttacact cagtattcca agcctttgtg
     3061 gcaggaagaa tagccactaa atggggcgaa aaaacggcag tactgctcgg atttattgca
     3121 gatagtagtg catttgcctt tttagcgttt atatctgaag gttggttagt tttccctgtt
     3181 ttaattttat tggctggtgg tgggatcgct ttacctgcat tacagggagt gatgtctatc
     3241 caaacaaaga gtcatcagca aggtgcttta cagggattat tggtgagcct taccaatgca
     3301 accggtgtta ttggcccatt actgtttgct gttatttata atcattcact accaatttgg
     3361 gatggctgga tttggattat tggtttagcg ttttactgta ttattatcct gctatcgatg
     3421 accttcatgt taacccctca agctcagggg agtaaacagg agacaagtgc ttagtgatcc
     3481 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3541 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcccgg ttaacgtgcc
     3601 ggcacggcct gggtaaccag gtattttgtc cacataaccg tgcgcaaaat gttgtggata
     3661 agcaggacac agcagcaatc cacagcaggc atacaaccgc acaccgaggt tactccgttc
     3721 tacaggttac gacgacatgt caatacttgc ccttgacagg cattgatgga atcgtagtct
     3781 cacgctgata gtctgatcga caatacaagt gggaccgtgg tcccagaccg ataatcagac
     3841 cgacaacacg agtgggatcg tggtcccaga ctaataatca gaccgacgat acgagtggga
     3901 ccgtggtccc agactaataa tcagaccgac gatacgagtg ggaccgtggt tccagactaa
     3961 taatcagacc gacgatacga gtgggaccgt ggtcccagac taataatcag accgacgata
     4021 cgagtgggac catggtccca gactaataat cagaccgacg atacgagtgg gaccgtggtc
     4081 ccagtctgat tatcagaccg acgatacgag tgggaccgtg gtcccagact aataatcaga
     4141 ccgacgatac gagtgggacc gtggtcccag actaataatc agaccgacga tacgagtggg
     4201 accgtggtcc cagtctgatt atcagaccga cgatacaagt ggaacagtgg gcccagagag
     4261 aatattcagg ccagttatgc tttctggcct gtaacaaagg acattaagta aagacagata
     4321 aacgtagact aaaacgtggt cgcatcaggg tgctggcttt tcaagttcct taagaatggc
     4381 ctcaattttc tctatacact cagttggaac acgagacctg tccaggttaa gcaccatttt
     4441 atcgccctta tacaatactg tcgctccagg agcaaactga tgtcgtgagc ttaaactagt
     4501 tcttgatgca gatgacgttt taagcacaga agttaaaaga gtgataactt cttcagcttc
     4561 aaatatcacc ccagcttttt tctgctcatg aaggttagat gcctgctgct taagtaattc
     4621 ctctttatct gcaaaggctt tttgaagtgc atcacctgac cgggcagata gttcaccggg
     4681 gtgagaaaaa agagcaacaa ctgatttagg caatttggcg gtgttgatac agcgggtaat
     4741 aatcttacgt gaaatatttt ccgcatcagc cagcgcagaa atatttccag caaattcatt
     4801 ctgcaatcgg cttgcataac gctgaccacg ttcataagca cttgttgggc gataatcgtt
     4861 acccaatctg gataatgcag ccatctgctc atcatccagc tcgccaacca gaacacgata
     4921 atcactttcg gtaagtgcag cagctttacg acggcgactc ccatcggcaa tttctatgac
     4981 accagatact cttcgaccga acgccggtgt ctgttgacca gtcagtagaa aagaagggat
     5041 gagatcatcc agtgcgtcct cagtaagcag ctcctggtca cgttcattac ctgaccatac
     5101 ccgagaggtc ttctcaacac tatcaccccg gagcacttca agagtaaact tcacatcccg
     5161 accacataca ggcaaagtaa tggcattacc gcgagccatt actcctacgc gcgcaattaa
     5221 cgaatccacc atcggggcag ctggtgtcga taacgaagta tcttcaaccg gttgagtatt
     5281 gagcgtatgt tttggaataa caggcgcacg cttcattatc taatctccca gcgtggttta
     5341 atcagacgat cgaaaatttc attgcagaca ggttcccaaa tagaaagagc atttctccag
     5401 gcaccagttg aagagcgttg atcaatggcc tgttcaaaaa cagttctcat ccggatcgga
     5461 cctttaccaa cttcatccgt ttcacgtaca acatttttta gaaccatgct tccccaggca
     5521 tcccgaattt gctcctccat ccacggggac tgagagccat tactattgct gtatttggta
     5581 agcaaaatac gtacatcagg ctcgaaccct ttaagatcaa cgttcttgag cagatcacga
     5641 agcatatcga aaaactgcag tgcggaggtg tagtcaaaca actcagcagg cgtgggaaca
     5701 atcagcacat cagcagcaca tacgacatta atcgtgccga tacccaggtt aggcgcgctg
     5761 tcaataacta tgacatcata gtcatgagca acagtttcaa tggccagtcg gagcatcagg
     5821 tgtggatcgg tgggcagttt accttcatca aatttgccca ttaactcagt ttcaatacgg
     5881 tgcagagcca gacaggaagg aataatgtca agccccggcc agcaagtggg ctttattgca
     5941 taagtgacat cgtccttttc cccaagatag aaaggcagga gagtgtcttc tgcatgaata
     6001 tgaagatctg gtacccatcc gtgatacatt gaggctgttc cctgggggtc gttaccttcc
     6061 acgagcaaaa cacgtagccc cttcagagcc agatcctgag caagatgaac agaaactgag
     6121 gttttgtaaa cgccaccttt atgggcagca accccgatca ccggtggaaa tacgtcttca
     6181 gcacgtcgca atcgcgtacc aaacacatca cgcatatgat taatttgttc aattgtataa
     6241 ccaacacgtt gctcaacccg tcctcgaatt tccatatccg ggtgcggtag tcgccctgct
     6301 ttctcggcat ctctgatagc ctgagaagaa accccaacta aatccgctgc ttcacctatt
     6361 ctccagcgcc gggttatttt cctcgcttcc gggctgtcat cattaaactg tgcaatggcg
     6421 atagccttcg tcatttcatg accagcgttt atgcactggt taagtgtttc catgagtttc
     6481 attctgaaca tcctttaatc attgctttgc gtttttttat taaatcttgc aatttactgc
     6541 aaagcaacaa caaaatcgca aagtcatcaa aaaaccgcaa agttgtttaa aataagagca
     6601 acactacaaa aggagataag aagagcacat acctcagtca cttattatca ctagcgctcg
     6661 ccgcagccgt gtaaccgagc atagcgagcg aactggcgag gaagcaaaga agaactgttc
     6721 tgtcagatag ctcttacgct cagcgcaaga agaaatatcc accgtgggaa aaactccagg
     6781 tagaggtaca cacgcggata gccaattcag agtaataaac tgtgataatc aaccctcatc
     6841 aatgatgacg aactaacccc cgatatcagg tcacatgacg aagggaaaga gaaggaaatc
     6901 aactgtgaca aactgccctc aaatttggct tccttaaaaa ttacagttca aaaagtatga
     6961 gaaaatccat gcaggctgaa ggaaacagca aaactgtgac aaattaccct cagtaggtca
     7021 gaacaaatgt gacgaaccac cctcaaatct gtgacagata accctcagac tatcctgtcg
     7081 tcatggaagt gatatcgcgg aaggaaaata cgatatgagt cgtctggcgg cctttctttt
     7141 tctcaatgta tgagaggcgc attggagttc tgctgttgat ctcattaaca cagacctgca
     7201 ggaagcggcg gcggaagtca ggcatacgct ggtaactttg aggcagctgg taacgctcta
     7261 tgatccagtc gattttcaga gagacgatgc ctgagccatc cggcttacga tactgacaca
     7321 gggattcgta taaacgcatg gcatacggat tggtgatttc ttttgtttca ctaagccgaa
     7381 actgcgtaaa ccggttctgt aacccgataa agaagggaat gagatatggg ttgatatgta
     7441 cactgtaaag ccctctggat ggactgtgcg cacgtttgat aaaccaagga aaagattcat
     7501 agcctatttc atcgccggca tcctcttcag ggcgataaaa aaccacttcc ttccccgcga
     7561 aactcttcaa tgcctgccgt atatccttac tggcttccgc agaggtcaat ccgaatattt
     7621 cagcatattt agcaacatgg atctcgcaga taccgtcatg ttcctgtagg gtgccatcag
     7681 attttctgat ctggtcaacg aacagataca gcatacgttt ttgatcccgg gagagactat
     7741 atgccgcctc agtgaggtcg tttgactgga cgattcgcgg gctattttta cgtttcttgt
     7801 gattgataac cgctgtttcc gccatgacag atccatgtga agtgtgacaa gtttttagat
     7861 tgtcacacta aataaaaaag agtcaataag cagggataac tttgtgaaaa aacagcttct
     7921 tctgagggca atttgtcaca gggttaaggg caatttgtca cagacaggac tgtcatttga
     7981 gggtgatttg tcacactgaa agggcaattt gtcacaacac cttctctaga accagcatgg
     8041 ataaaggcct acaaggcgct ctaaaaaaga agatctaaaa actataaaaa aaataattat
     8101 aaaaatatcc ccgtggataa gtggataacc ccaagggaag ttttttcagg catcgtgtgt
     8161 aagcagaata tataagtgct gttccctggt gcttcctcgc tcactcgagc tacggggtct
     8221 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     8281 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     8341 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     8401 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     8461 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     8521 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     8581 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     8641 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     8701 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     8761 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     8821 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     8881 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     8941 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     9001 agcagaactt taaaagtgct catcattgga aaacgt
//
