LOCUS       sequence-328839-        3759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For purification of mCerulean3 fluorophore.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3759)
  AUTHORS   Osterlund EJ, Hirmiz N, Pemberton JM, Nougarede A, Liu Q, Leber B, 
            Fang Q, Andrews DW
  TITLE     Efficacy and specificity of inhibitors of BCL-2 family protein 
            interactions assessed by affinity measurements in live cells.
  JOURNAL   Sci Adv. 2022 Apr 22;8(16):eabm7375. doi: 10.1126/sciadv.abm7375. 
            Epub 2022 Apr 20.
  PUBMED    35442739
REFERENCE   2  (bases 1 to 3759)
  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: "Sci Adv."; 
            date: "2022-04-22"; volume: "8(16):eabm7375. doi"; pages: " 
            10.1126/sciadv.abm7375. Epub 2022 Apr 20"
COMMENT     Sequence Label: His6-TEV-mCerulean3-pBluescript
FEATURES             Location/Qualifiers
     source          1..3759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(37..465)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(147..168)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     359..378
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     592..614
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     606..623
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     607..623
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             693..715
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             726..743
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             771..791
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(795..797,798..800,801..1514)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(840..861)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1101..1120)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1448..1469
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1578..1596)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1617..1633)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1617..1633)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1630..1652)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1641..1657
                     /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)."
     promoter        complement(join(1665..1671,1672..1689,1690..1695))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1710..1731
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1848..1865)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2019..2607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2099..2118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2778..3569,3570..3638))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3401..3420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3639..3743)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
       61 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
      121 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
      181 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
      241 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
      301 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
      361 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
      421 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca
      481 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
      541 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
      601 gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc gaattggagc
      661 tccaccgcgg tggcggccgc tctagaaata attttgttta actttaagaa ggagatataa
      721 ccatgcatca ccaccaccat cacggcggca gcggcggtac gggcggtagc gagaacttat
      781 actttcaagg caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      841 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      901 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      961 tgccctggcc caccctcgtg accaccctga gctggggcgt gcagtgcttc gcccgctacc
     1021 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1081 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1141 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1201 acatcctggg gcacaagctg gagtacaacg ccatccacgg caacgtctat atcaccgccg
     1261 acaagcagaa gaacggcatc aaggccaact tcggcctcaa ctgcaacatc gaggacggca
     1321 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1381 tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc
     1441 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1501 agctgtacaa gtaaggactc agatccacgc gtatcgattg tcgacctcga gggggggccc
     1561 ggtaccagct tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca
     1621 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     1681 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     1741 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1801 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     1861 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1921 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     1981 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2041 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2101 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2161 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2221 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2281 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2341 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2401 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     2461 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2521 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2581 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2641 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2701 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2761 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2821 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2881 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     2941 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3001 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3061 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3121 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3181 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3241 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3301 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3361 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3421 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3481 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3541 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3601 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3661 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3721 aataaacaaa taggggttcc gcgcacattt ccccgaaaa
//
