LOCUS       sequence-401165-        6769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6769)
  AUTHORS   Evers TH, van Dongen EM, Faesen AC, Meijer EW, Merkx M
  TITLE     Quantitative understanding of the energy transfer between 
            fluorescent proteins connected via flexible peptide linkers.
  JOURNAL   Biochemistry. 2006 Nov 7. 45(44):13183-92.
  PUBMED    17073440
REFERENCE   2  (bases 1 to 6769)
  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: 
            "Biochemistry. 2006 Nov 7. 45(44):13183-92."
COMMENT     Sequence Label: pET28CLY1
FEATURES             Location/Qualifiers
     source          1..6769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     97..116
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     200..219
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        200..218
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    219..243
                     /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             258..280
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             299..316
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             326..343
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(347..349,350..352,353..1063)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1097..1099,1100..1102,1103..1816)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1833..1850
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1903..1921)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1917..1964
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2001..2456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2088..2107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2298..2319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2548..3363)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2548..3363)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3271..3290
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3355..3374
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3485..4073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3974..3993
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4227..4244
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4259..4398
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4324..4343)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4484..4506
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4500..4691)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5500..6582)
                     /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     6544..6563
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6583..6660)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
       61 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
      121 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
      181 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
      241 tcccctctag aaataatttt gtttaacttt aagaaggaga tatactatgg gcagcagcca
      301 tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatgg tgagcaaggg
      361 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      421 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
      481 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
      541 gacctggggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
      601 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
      661 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
      721 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
      781 ctacatcagc cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa
      841 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
      901 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
      961 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1021 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggaa ttcgtggtgg
     1081 atccggaggt agcaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1141 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1201 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1261 cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgcagtgct tcgcccgcta
     1321 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1381 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1441 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1501 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1561 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1621 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1681 gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa
     1741 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1801 cgagctgtac aagtaaagcg gccgcactcg agcaccacca ccaccaccac tgagatccgg
     1861 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     1921 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     1981 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2041 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2101 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2161 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2221 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2281 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2341 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2401 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     2461 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2521 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg
     2581 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     2641 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     2701 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     2761 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     2821 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     2881 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     2941 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     3001 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     3061 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     3121 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     3181 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     3241 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     3301 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
     3361 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
     3421 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3481 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3541 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3601 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3661 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3721 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3781 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3841 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3901 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3961 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4021 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4081 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4141 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4201 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4261 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     4321 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     4381 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4441 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4501 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4561 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4621 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4681 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4741 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4801 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4861 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4921 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4981 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     5041 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     5101 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     5161 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     5221 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     5281 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     5341 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     5401 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     5461 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5521 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5581 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     5641 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     5701 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     5761 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     5821 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     5881 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     5941 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     6001 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     6061 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     6121 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     6181 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     6241 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     6301 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     6361 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     6421 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     6481 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     6541 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     6601 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     6661 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     6721 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagat
//
