LOCUS       sequence-14304-e        7145 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 7145)
  AUTHORS   Luker KE, Smith MC, Luker GD, Gammon ST, Piwnica-Worms H, 
            Piwnica-Worms D
  TITLE     Kinetics of regulated protein-protein interactions revealed with 
            firefly luciferase complementation imaging in cells and living 
            animals.
  JOURNAL   Proc Natl Acad Sci U S A. 2004 Aug 17. 101(33):12288-93.
  PUBMED    15284440
REFERENCE   2  (bases 1 to 7145)
  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: "Proc Natl 
            Acad Sci U S A. 2004 Aug 17. 101(33):12288-93."
COMMENT     Sequence Label: FRB-NLuc
FEATURES             Location/Qualifiers
     source          1..7145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             965..1234
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     primer_bind     complement(1343..1361)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             2642..2683
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2693..2710
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2733..2750)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2739..2963
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3009..3437
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3096..3115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3306..3327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3446..3466)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3451..3781
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3632..3767
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3694..3713
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3848..4642
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3902..3921)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4512..4531
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4818..4939
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4855..4874)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4909..4928
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4988..5004)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4988..5004)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5001..5023)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5012..5028
                     /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(5036..5042,5043..5060,5061..5066))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5081..5102
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5219..5236)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5390..5978)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5470..5489)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6149..6940,6941..7009))
                     /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     6772..6791
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7010..7114)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttggt accgagctcg gatccactag tccagtgtgg tggaattgcc cttacaccac
      961 catggagatg tggcatgaag gcctggaaga ggcatctcgt ttgtactttg gggaaaggaa
     1021 cgtgaaaggc atgtttgagg tgctggagcc cttgcatgct atgatggaac ggggccccca
     1081 gactctgaag gaaacatcct ttaatcaggc ctatggtcga gatttaatgg aggcccaaga
     1141 gtggtgcagg aagtacatga aatcagggaa tgtcaaggac ctcacccaag cctgggacct
     1201 ctattatcat gtgttccgac gaatctcaaa gcagcagatc tcgtacgcgt cccggggcgg
     1261 tggctcatct ggcggaggtg aagacgccaa aaacataaag aaaggcccgg cgccattcta
     1321 tccgctggaa gatggaaccg ctggagagca actgcataag gctatgaaga gatacgccct
     1381 ggttcctgga acaattgctt ttacagatgc acatatcgag gtggacatca cttacgctga
     1441 gtacttcgaa atgtccgttc ggttggcaga agctatgaaa cgatatgggc tgaatacaaa
     1501 tcacagaatc gtcgtatgca gtgaaaactc tcttcaattc tttatgccgg tgttgggcgc
     1561 gttatttatc ggagttgcag ttgcgcccgc gaacgacatt tataatgaac gtgaattgct
     1621 caacagtatg ggcatttcgc agcctaccgt ggtgttcgtt tccaaaaagg ggttgcaaaa
     1681 aattttgaac gtgcaaaaaa agctcccaat catccaaaaa attattatca tggattctaa
     1741 aacggattac cagggatttc agtcgatgta cacgttcgtc acatctcatc tacctcccgg
     1801 ttttaatgaa tacgattttg tgccagagtc cttcgatagg gacaagacaa ttgcactgat
     1861 catgaactcc tctggatcta ctggtctgcc taaaggtgtc gctctgcctc atagaactgc
     1921 ctgcgtgaga ttctcgcatg ccagagatcc tatttttggc aatcaaatca ttccggatac
     1981 tgcgatttta agtgttgttc cattccatca cggttttgga atgtttacta cactcggata
     2041 tttgatatgt ggatttcgag tcgtcttaat gtatagattt gaagaagagc tgtttctgag
     2101 gagccttcag gattacaaga ttcaaagtgc gctgctggtg ccaaccctat tctccttctt
     2161 cgccaaaagc actctgattg acaaatacga tttatctaat ttacacgaaa ttgcttctgg
     2221 tggcgctccc ctctctaagg aagtcgggga agcggttgcc aagaggttcc atctgccagg
     2281 tatcaggcaa ggatatgggc tcactgagac tacatcagct attctgatta cacccgaggg
     2341 ggatgataaa ccgggcgcgg tcggtaaagt tgttccattt tttgaagcga aggttgtgga
     2401 tctggatacc gggaaaacgc tgggcgttaa tcaaagaggc gaactgtgtg tgagaggtcc
     2461 tatgattatg tccggttatg taaacaatcc ggaagcgacc aacgccttga ttgacaagga
     2521 tggatgataa cattctggta gctaggtaat gcataactag ctaaggtacc caattaaggg
     2581 caattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgcggttcga
     2641 aggtaagcct atccctaacc ctctcctcgg tctcgattct acgcgtaccg gtcatcatca
     2701 ccatcaccat tgagtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     2761 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2821 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2881 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2941 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3001 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3061 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3121 tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt
     3181 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3241 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3301 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3361 tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca
     3421 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3481 ggctccccag gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3541 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3601 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3661 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3721 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3781 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc
     3841 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     3901 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     3961 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     4021 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     4081 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     4141 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     4201 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     4261 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     4321 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     4381 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     4441 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     4501 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     4561 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     4621 ccttcttgac gagttcttct gagcgggact ctggggttcg cgaaatgacc gaccaagcga
     4681 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     4741 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4801 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4861 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4921 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     4981 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5041 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5101 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5161 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5221 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5281 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5341 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5401 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5461 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5521 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5581 ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5641 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5701 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5761 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5821 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5881 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5941 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6001 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6061 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6121 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6181 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6241 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6301 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6361 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6421 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6481 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6541 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6601 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6661 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6721 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6781 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6841 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6901 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6961 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7021 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7081 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7141 acgtc
//
