LOCUS       sequence-55668-e        7318 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7318)
  AUTHORS   Parikh MR, Greene DN, Woods KK, Matsumura I
  TITLE     Directed evolution of RuBisCO hypermorphs through genetic selection 
            in engineered E.coli.
  JOURNAL   Protein Eng Des Sel. 2006 Mar;19(3):113-9. Epub 2006 Jan 19.
  PUBMED    16423843
REFERENCE   2  (bases 1 to 7318)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein Eng
            Des Sel."; date: "2006-03"; volume: "19(3)"; pages: "113-9. Epub 
            2006 Jan 19"
COMMENT     Sequence Label: rbcLS-pMAL-2px
FEATURES             Location/Qualifiers
     source          1..7318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1882..1899)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1882..1898)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1891..1913)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2077..2094)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2247..2333
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2425..2452
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2472..2563
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2564..2632,2633..3424)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2782..2801)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3466..3979)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3661..3682)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3873..3892
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4090..4678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4579..4598
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4832..4849
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4864..5006
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4932..4951)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5092..5114
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5108..5299)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5795..5872
                     /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             5873..6955
                     /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(5892..5911)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(7198..7203,7204..7219,7220..7226)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    7234..7250
                     /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     7239..7261
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 tatgcccaag acgcaatctg ccgcaggcta taaggccggg gtgaaggact acaaactcac
       61 ctattacacc cccgattaca cccccaaaga cactgacctg ctggcggctt tccgcttcag
      121 ccctcagccg ggtgtccctg ctgacgaagc tggtgcggcg atcgcggctg aatcttcgac
      181 cggtacctgg accaccgtgt ggaccgactt gctgaccgac atggatcggt acaaaggcaa
      241 gtgctaccac atcgagccgg tgcaaggcga agagaactcc tactttgcgt tcatcgctta
      301 cccgctcgac ctgtttgaag aagggtcggt caccaacatc ctgacctcga tcgtcggtaa
      361 cgtgtttggc ttcaaagcta tccgttcgct gcgtctggaa gacatccgct tccccgtcgc
      421 cttggtcaaa accttccaag gtcctcccca cggtatccaa gtcgagcgcg acctgctgaa
      481 caagtacggc cgtccgatgc tgggttgcac gatcaaacca aaactcggtc tgtcggcgaa
      541 aaactacggt cgtgccgtct acgaatgtct gcgcggcggt ctggacttca ccaaagacga
      601 cgaaaacatc aactcgcagc cgttccaacg ctggcgcgat cgcttcctgt ttgtggctga
      661 tgcaatccac aaatcgcaag cagaaaccgg tgaaatcaaa ggtcactacc tgaacgtgac
      721 cgcgccgacc tgcgaagaaa tgatgaaacg ggctgagttc gctaaagaac tcggcatgcc
      781 gatcatcatg catgacttct tgacggctgg tttcaccgcc aacaccacct tggcaaaatg
      841 gtgccgcgac aacggcgtcc tgctgcacat ccaccgtgca atgcacgcgg tgatcgaccg
      901 tcagcgtaac cacgggattc acttccgtgt cttggccaag tgtttgcgtc tgtccggtgg
      961 tgaccacctc cactccggca ccgtcgtcgg caaactggaa ggcgacaaag cttcgacctt
     1021 gggctttgtt gacttgatgc gcgaagacca catcgaagct gaccgcagcc gtggggtctt
     1081 cttcacccaa gattgggcgt cgatgccggg cgtgctgccg gttgcttccg gtggtatcca
     1141 cgtgtggcac atgcccgcac tggtggaaat cttcggtgat gactccgttc tccagttcgg
     1201 tggcggcacc ttgggtcacc cctggggtaa tgctcctggt gcaaccgcga accgtgttgc
     1261 cttggaagct tgcgtccaag ctcggaacga aggtcgcgac ctctaccgtg aaggcggcga
     1321 catccttcgt gaagctggca agtggtcgcc tgaactggct gctgccctcg acctctggaa
     1381 agagatcaag ttcgaattcg aaacgatgga caagctctaa ggagcctctg actatcgctg
     1441 ggggagtgag cgttgctgcg taaagctttc tccccagcct ttcgacttaa cctttcagga
     1501 tttctgaatc atgagcatga aaactctgcc caaagagcgt cgtttcgaga ctttctcgta
     1561 cctgcctccc ctcagcgatc gccaaatcgc tgcacaaatc gagtacatga tcgagcaagg
     1621 cttccacccc ttgatcgagt tcaacgagca ctcgaatccg gaagagttct actggacgat
     1681 gtggaagctc cccctgtttg actgcaagag ccctcagcaa gtcctcgatg aagtgcgtga
     1741 gtgccgcagc gaatacggtg attgctacat ccgtgtcgct ggcttcgaca acatcaagca
     1801 gtgccaaacc gtgagcttca tcgttcatcg tcccggccgc tactaaagcc tgagctccgt
     1861 cgacctgcag gcaagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     1921 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     1981 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggca
     2041 gcttggctgt tttggcggat gagataagat tttcagcctg atacagatta aatcagaacg
     2101 cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga
     2161 ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca
     2221 tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     2281 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg
     2341 gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat
     2401 aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc
     2461 tacaaactct ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2521 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2581 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2641 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2701 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttctccaat
     2761 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca
     2821 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2881 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2941 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3001 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3061 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3121 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3181 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3241 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     3301 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3361 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3421 gtaactgtca gaccaagttt actcatatat actttagatt gatttacccc ggttgataat
     3481 cagaaaagcc ccaaaaacag gaagattgta taagcaaata tttaaattgt aaacgttaat
     3541 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     3601 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     3661 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     3721 accgtctatc agggcgatgg cccactacgt gaaccatcac ccaaatcaag ttttttgggg
     3781 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     3841 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     3901 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     3961 gcgccgctac agggcgcgta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4021 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4081 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4141 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4201 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4261 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4321 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4381 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4441 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4501 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4561 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4621 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4681 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4741 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4801 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4861 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg
     4921 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4981 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     5041 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5101 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     5161 catcagcgtg gtcgtgcagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     5221 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     5281 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     5341 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     5401 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     5461 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     5521 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     5581 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5641 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5701 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     5761 cccgtggcca ggacccaacg ctgcccgaaa ttccgacacc atcgaatggt gcaaaacctt
     5821 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     5881 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     5941 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     6001 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     6061 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     6121 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     6181 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     6241 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     6301 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     6361 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     6421 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     6481 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     6541 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     6601 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     6661 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     6721 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     6781 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     6841 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6901 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     6961 acgcaattaa tgtaagttag ctcactcatt aggcacaatt ctcatgtttg acagcttatc
     7021 atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg
     7081 gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg
     7141 ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg
     7201 acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag
     7261 gaaacagcca gtccgtttag gtgttttcac gagcacttca ccaacaagga ccatagca
//
