LOCUS       sequence-7679-ex        5289 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 5289)
  AUTHORS   Gu H, Lalonde S, Okumoto S, Looger LL, Scharff-Poulsen AM, Grossman 
            AR, Kossmann J, Jakobsen I, Frommer WB
  TITLE     A novel analytical method for in vivo phosphate tracking.
  JOURNAL   FEBS Lett. 2006 Oct 30. 580(25):5885-93.
  PUBMED    17034793
REFERENCE   2  (bases 1 to 5289)
  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: "FEBS Lett. 
            2006 Oct 30. 580(25):5885-93."
COMMENT     Sequence Label: pRSET FLIPPi-4u
FEATURES             Location/Qualifiers
     source          1..5289
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             112..129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             133..165
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(169..177,178..192)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(205..207,208..210,211..723)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIE"
     CDS             join(1915..1917,1918..1920,1921..2634)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2690..2708)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2704..2751
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2848..3303
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2935..2954)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3145..3166
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3329..3433
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3434..3502,3503..4294)
                     /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     complement(3652..3671)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4465..5053
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4954..4973
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5207..5224
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat
      121 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac
      181 gatgacgata aggatccggg ccgcatggtg agcaagggcg aggagctgtt caccggggtg
      241 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
      301 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
      361 aagctgcccg tgccctggcc caccctcgtg accaccctga cctggggcgt gcagtgcttc
      421 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
      481 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
      541 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
      601 gaggacggca acatcctggg gcacaagctg gagtacaact acatcagcca caacgtctat
      661 atcaccgccg acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc
      721 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
      781 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
      841 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcggtacc
      901 gtaggatttc taacagcgac ctcggctcaa gcccaaaccg tgcaaatctc cggggcgggc
      961 gcgacctttg cggctccttt gctgcaacgt tggtttgacg cctacaaccg caccgtagac
     1021 cccactgtgc aagtcagcta tcagtctgtc ggtagtggtg ctggcctaga gcaggtgatc
     1081 aatggcactg tggacttcgg cgcttccgag gcgcctttct ccggtgctcg cctggagagc
     1141 ttccgagcta aatacggcta tgatccccta cagttgcctc tggcgggagg ggccatcgag
     1201 tttgcctata acctgcccgg cattgaagac ggagagctca tcctgaagcg gaaaacctac
     1261 tgcggcatcg tgaccggcga gatcactcgc tgggacgaca ttcgcatcaa ggccgagaac
     1321 ccaggtatag caaacaagct gccacccctg gacatcacct gggtacaccg ctctgatggt
     1381 tctggggcta cctttgtgtt caccaaccac atcagaactg tctgccctaa ttggacagcc
     1441 ggtgctggta cttctgtcga gtggcctgtt ggtattggag cccaagggaa tgagggcgta
     1501 gccgccacca tcaagcagga gccaggggcg attggctacg tgaaccagtc ctatgccaag
     1561 ctggaaaaga tggccactgc tcgcttggaa aacaaagcgg gcaacattgt tgagttctcg
     1621 actgaggcag ctacctcggc gctggatgct cccattcctg atgactttgc gctgttggtg
     1681 cccgaccctg aagggccaaa tgactaccca atcgtgggct tgttctgggt gatgctgtac
     1741 cgcgagtatc ccgatcagca gaagctgacc aagctggtgg aggctctgaa gtggacccag
     1801 gggccagagg gtcaagccat caccaaggag ctggactaca tccctatgcc tgaggcggtt
     1861 atccagcgga tctttgcaga gctggattcc atcaccgtta acggtaccgg tggaatggtg
     1921 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1981 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     2041 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     2101 accaccttcg gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     2161 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     2221 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2281 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2341 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     2401 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     2461 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2521 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2581 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaaagctt
     2641 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     2701 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     2761 ggaactatat ccggatctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2821 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     2881 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2941 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3001 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3061 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3121 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3181 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3241 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     3301 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3361 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3421 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     3481 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3541 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3601 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3661 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3721 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3781 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3841 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3901 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3961 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     4021 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     4081 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     4141 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     4201 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     4261 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     4321 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     4381 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4441 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4501 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4561 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4621 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4681 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4741 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4801 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4861 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4921 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4981 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5041 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5101 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5161 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5221 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     5281 ttaatgcag
//
