LOCUS       sequence-477888-        5704 bp    DNA     circular SYN 08-JUL-2025
DEFINITION  High copy vector for expression of GWS1B Rubisco.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5704)
  AUTHORS   McDonald JL, Shapiro NP,  Whitney SM, Wilson RH, Shoulders MD
  TITLE     In Vivo Directed Evolution of an Ultra-Fast Rubisco from a 
            Semi-Anaerobic Environment Imparts Oxygen Resistance
  JOURNAL   bioRxiv 2025.02.17.638297
REFERENCE   2  (bases 1 to 5704)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.02.17.638297"
COMMENT     Sequence Label: pTrc-GWS1B
FEATURES             Location/Qualifiers
     source          1..5704
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(841..860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1001..1023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        1101..1178
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             1179..2261
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1198..1217)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2493..2498,2499..2515,2516..2522)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2530..2546
                     /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)."
     primer_bind     2535..2557
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    2563..2632
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             2683..2709
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             4090..4107
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4188..4205)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4358..4444
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4536..4563
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4582..4673
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4674..4742,4743..5534)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4892..4911)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
      841 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      901 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      961 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1021 aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg
     1081 aagcggcatg catttacgtt gacaccatcg aatggtgcaa aacctttcgc ggtatggcat
     1141 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     1201 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     1261 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     1321 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     1381 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     1441 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     1501 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     1561 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     1621 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     1681 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     1741 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     1801 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     1861 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     1921 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     1981 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     2041 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     2101 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     2161 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     2221 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta
     2281 agttagcgcg aattgatctg gtttgacagc ttatcatcga ctgcacggtg caccaatgct
     2341 tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca
     2401 taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat
     2461 aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatccg gctcgtataa
     2521 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagcgccgct gagaaaaagc
     2581 gaagcggcac tgctctttaa caatttatca gacaatctgt gtgggcactc gaccggaatt
     2641 atcgattaac tttattatta aaaattaaag aggtatatat taatgtatcg attaaataag
     2701 gaggaataaa ccatggacca aagtaatcgt tacgccgact tatccttgaa agaggaggac
     2761 ttgatcaagg gggggaatca tatcctggtt gcctatacaa tggaacccgc tgccggggtc
     2821 ggatatcttg aagcagccgc ccacattgcc gcagagagtt ctactggaac caatgtggag
     2881 gtctcgacta cggatgaatt tacaaaaggg gtcgatgctc ttgtgtattt cattgatgag
     2941 gccaagggca tcatgaaggt cgcgtatcct aatgatctgt tcgatcgcaa tgttaccgat
     3001 gggcgcgtga tgatcgtttc cttcttgacc ctttgtattg gaaataatca aggaatggga
     3061 gacattgcaa atctgcagat gcaggacttc tacgttcccc cgcgtatgtt gcaactgttt
     3121 gacgggcctg cgaaggatat ttcagattta tggcgcatct tgggtcgtcc agttaaagat
     3181 ggtgggtaca tctcaggaac aattattaaa ccgaaattag gtcttcgtcc cgaacctttc
     3241 gctaaggcag cctatcaatt ttggcttggc ggtgatttta tcaagaatga tgagccccaa
     3301 ggtaaccagg tattttgccc aatcaagaaa gtcctgccct tggtttacga ttcgttaaag
     3361 cgcgcccagg acgaaactgg gcaagccaaa ttgtttagta tgaacatcac ggcagatgat
     3421 cattacgaga tgtgcgcgcg cgctgacttt gcccttgaaa ccttcgggcc ggatgctgac
     3481 aaagtggcct ttcttgttga cggttttgtc ggtgggccgg ggatggttac aacagcccgt
     3541 cgccaatatc cctcccaata cttgcactac caccgtgcag gtcacggcat ggtgactagc
     3601 ccatcctcta agcgcggata cacggccttt gtgctggcga agatgagtcg tttacaaggg
     3661 gcatccggga tccatgttgg cactatggga tacggaaaaa tggaaggcgg caaagacgat
     3721 cgcattattg cctacatgat cgagcgtgat agctgcacgg gaccattcta tcatcaagag
     3781 tggtatggta tgaagcctac aactccaatt atttctggag gtatgaacgc tttgcgtttg
     3841 cctggcttct tcgagaatct gggacacgga aacgtaatta acactgctgg cggtggaagc
     3901 tacggccata tcgactcgcc agcggcagga gctgtctcgt tacgccaggc ctatgaatgt
     3961 tggaaagctg gggccgatcc tatcgagtgg gctaaggagc acaaggaatt cgcgcgtgcg
     4021 tttgaatcct ttccaaaaga cgcggacaaa ttgttccccg gatggcgcga aaaattagga
     4081 gtacataaac accaccacca ccaccactag tggtttaatt aacgcagcct gaatggcgaa
     4141 taggagctca agcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt
     4201 aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg
     4261 gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg
     4321 gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
     4381 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     4441 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
     4501 acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct
     4561 ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc
     4621 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4681 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4741 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4801 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4861 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     4921 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4981 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5041 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5101 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5161 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5221 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5281 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5341 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5401 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5461 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5521 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5581 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5641 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5701 cttc
//
