LOCUS       sequence-126607-        6148 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for Cerulean purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6148)
  AUTHORS   Niederholtmeyer H, Sun ZZ, Hori Y, Yeung E, Verpoorte A, Murray RM, 
            Maerkl SJ
  TITLE     Rapid cell-free forward engineering of novel genetic ring 
            oscillators.
  JOURNAL   Elife. 2015 Oct 2;4. doi: 10.7554/eLife.09771.
  PUBMED    26430766
REFERENCE   2  (bases 1 to 6148)
  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: "Elife."; 
            date: "2015-10-2"; volume: "4. doi"; pages: " 10.7554/eLife.09771"
COMMENT     Sequence Label: pET21a(+)-Histag-Cerulean (526)
FEATURES             Location/Qualifiers
     source          1..6148
                     /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             101..118
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             125..838
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             125..838
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(155..183)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     778..798
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             876..893
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(946..964)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      960..1007
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1044..1499
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1131..1150)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1341..1362
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1526..1630
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1631..1699,1700..2491)
                     /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(1849..1868)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2662..3250
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3151..3170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3404..3421
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3436..3578
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3504..3523)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3664..3686
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3680..3871)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4680..5762)
                     /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     5724..5743
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5763..5840)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6046..6065
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gagaggatcg catcaccatc accatcacgg
      121 atccatgagt aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga
      181 tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata
      241 cggaaaactt acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac
      301 acttgtcact actttgactt ggggtgttca atgctttgct agatacccag atcatatgaa
      361 acagcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt
      421 tttcaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct
      481 tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca
      541 caaattggaa tacaacgcta tttcagataa tgtatacatc actgcagaca aacaaaagaa
      601 tggaatcaaa gctaatttca aaattagaca caacattgaa gatggaagcg ttcaactagc
      661 agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca
      721 ttacctgtcc acacaatctg ccctttcgaa agatcccaac gaaaagagag atcacatggt
      781 ccttcttgag tttgtaacag ctgctgggat tacactaggc atggatgaac tatacaaata
      841 agagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc
      901 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
      961 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1021 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1081 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1141 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1201 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1261 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1321 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1381 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1441 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     1501 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1561 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     1621 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     1681 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     1741 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     1801 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     1861 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     1921 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     1981 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2041 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2101 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2161 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     2221 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     2281 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     2341 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     2401 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     2461 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     2521 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     2581 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2641 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2701 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2761 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2821 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2881 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2941 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3001 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3061 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3121 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3181 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3241 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3301 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3361 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3421 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3481 ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3541 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     3601 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3661 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     3721 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     3781 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     3841 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     3901 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     3961 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     4021 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     4081 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     4141 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     4201 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     4261 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     4321 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     4381 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     4441 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     4501 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     4561 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     4621 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     4681 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4741 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     4801 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     4861 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     4921 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     4981 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5041 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     5101 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5161 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5221 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5281 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5341 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5401 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5461 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5521 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     5581 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     5641 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     5701 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     5761 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     5821 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     5881 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     5941 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     6001 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     6061 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     6121 gaggatcgag atctcgatcc cgcgaaat
//
