LOCUS       sequence-354358-        6488 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Inducible expression of UniProt identifier C2K1F0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6488)
  AUTHORS   Rembeza E, Engqvist MKM
  TITLE     Experimental and computational investigation of enzyme functional 
            annotations uncovers misannotation in the EC 1.1.3.15 enzyme class.
  JOURNAL   PLoS Comput Biol. 2021 Sep 23;17(9):e1009446. doi: 
            10.1371/journal.pcbi.1009446. eCollection 2021 Sep.
  PUBMED    34555022
REFERENCE   2  (bases 1 to 6488)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS Comput
            Biol."; date: "2021-09-23"; volume: "17(9):e1009446. doi"; pages: " 
            10.1371/journal.pcbi.1009446. eCollection 2021 Sep"
COMMENT     Sequence Label: C2K1F0
FEATURES             Location/Qualifiers
     source          1..6488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1208..1237
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1290..1308)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1304..1351
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1388..1843
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1475..1494)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1685..1706
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1869..1973
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1974..2042,2043..2834)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2192..2211)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3005..3593
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3494..3513
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3747..3764
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3779..3918
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3844..3863)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4004..4026
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4020..4211)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5020..6102)
                     /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     6064..6083
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6103..6180)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6386..6405
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat ggtggatgcg gtgaaggcgg acccgtttgg
      121 taaagtcaat gcgatcgatg tactggatct ggccagcctg gaagcacgtg cggagaaagt
      181 gttaggtcgc ggcgagttcg gctacatcag cgaaggtagc gatgacggct atacgatgca
      241 ccgcaacacc accgcctttc aggacgtaca tatgttacca cgtgtgttgc agggcgtgga
      301 gaacccagat cagtcaacca ccttcatggg cgccaaactt gcgtcaccgt tactgactgc
      361 tccaatcgct agcaacactc ttgcccatcc gagcggtgaa ctcgggttgg caaaaggggc
      421 gaaggaagcg ggtattatga tgagccaatc aacattcgcg agcaaaacca ttgcggagac
      481 cgccgcggtc agcgatggtg cgccgtatat gttccaactc tatatgccga aggattggga
      541 atactgccag tacctgttag atgaggcgaa acaggcaggt gccctggcaa tcattctgac
      601 agccgactcg accctgggcg gctatcgcga aaaggacgtt atgaaccatt accatttaaa
      661 aggccgcctt gccaacctgg aagggtataa tactggtcag agtggagtag gggccggtgg
      721 gttattcaaa gagtcgatgc agaaattgga cttaggactt atctcgaagc tggcaagtta
      781 ctccggtctg ccgattatta tcaaagggat ccaacatccg gccgacgctg tcgctgcgat
      841 tacggctgga gcggcaggaa tttacgtgtc gaaccatggc ggtcgtcagc tggatggtgc
      901 cccaggtgcg atcgaacagc tgccggcgat tgccgcggca gtggatcatc gtgtgcctat
      961 tatctttgat ggcggagttc agcgcggcac acatgttctg aaagcgctgg ccctgggtgc
     1021 ggacctggtt gggatcggac gcccgtttag ctatggttta gccctgggcg gttggcaagg
     1081 cgttaaagat gtcgctgacc acttgaagat ggagatcaat atcgcgatgc agctgaccgg
     1141 gtgccagacc atggcggacg tgaaacaaat gaaagttaaa acgaccttcg cagcggccgc
     1201 actcgagcat catcaccatc accaccacca ccaccactga gatccggctg ctaacaaagc
     1261 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1321 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     1381 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1441 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1501 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1561 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     1621 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     1681 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     1741 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     1801 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     1861 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1921 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1981 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2041 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2101 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2161 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     2221 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2281 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2341 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2401 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     2461 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     2521 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     2581 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2641 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2701 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2761 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2821 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     2881 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     2941 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3001 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3061 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3121 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3181 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3241 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3301 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3361 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3421 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3481 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3541 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3601 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3661 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3721 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3781 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     3841 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3901 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3961 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4021 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4081 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4141 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4201 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4261 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4321 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4381 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4441 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4501 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4561 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4621 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     4681 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     4741 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     4801 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     4861 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     4921 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     4981 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5041 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5101 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     5161 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     5221 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     5281 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     5341 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     5401 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     5461 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     5521 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     5581 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     5641 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     5701 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     5761 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     5821 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     5881 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     5941 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     6001 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     6061 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     6121 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     6181 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     6241 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     6301 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     6361 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     6421 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     6481 cgcgaaat
//
