LOCUS       sequence-199614-        4398 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  create your own 2in1 constructs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4398)
  AUTHORS   Grefen C, Blatt MR
  TITLE     A 2in1 cloning system enables ratiometric bimolecular fluorescence 
            complementation (rBiFC).
  JOURNAL   Biotechniques. 2012 Oct 2;53(5):311-14. doi: 10.2144/000113941.
  PUBMED    23066669
REFERENCE   2  (bases 1 to 4398)
  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"; doi: "10"; journalName: 
            "Biotechniques"; date: "2012-10-2- 2"; volume: "53"; issue: "5"; 
            pages: "311-14"
COMMENT     Sequence Label: pUC57-R1R4
FEATURES             Location/Qualifiers
     source          1..4398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    414..538
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(563..568,569..586,587..593)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             647..1306
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(713..732)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1648..1953
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1832..1851
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1994..2118)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2177..2193)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2177..2193)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2190..2212)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2201..2217
                     /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(2225..2231,2232..2249,2250..2255))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2270..2291
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2408..2425)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2579..3167)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2659..2678)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3338..4129,4130..4198))
                     /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     3961..3980
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4199..4303)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4371..4389
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cactagtcct agaacaagtt
      421 tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga
      481 ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag tcactatggc
      541 ggccgcatta ggcaccccag gctttacact ttatgcttcc ggctcgtata atgtgtggat
      601 tttgagttag gatccgtcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat
      661 cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt
      721 tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt
      781 aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg
      841 cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg
      901 ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct
      961 ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc
     1021 gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt
     1081 ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa
     1141 cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat
     1201 gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct
     1261 taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaaagat ctggatccgg
     1321 cttactaaaa gccagataac agtatgcgta tttgcgcgct gatttttgcg gtataagaat
     1381 atatactgat atgtataccc gaagtatgtc aaaaagaggt atgctatgaa gcagcgtatt
     1441 acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct
     1501 ccggtctggt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga
     1561 aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt tattgaaatg aacggctctt
     1621 ttgctgacga gaacaggggc tggtgaaatg cagtttaagg tttacaccta taaaagagag
     1681 agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg
     1741 atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac
     1801 ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg
     1861 ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa
     1921 aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat acacagccag
     1981 tctgcaggtc gaccatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt
     2041 tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt ctcgttcaac
     2101 ttttctatac aaagttggtc aattgttata aatcggatcc cgggcccgtc gactgcagaa
     2161 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2221 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2281 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2341 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2401 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2461 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2521 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2581 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2641 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2701 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2761 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2821 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2881 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2941 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3001 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3061 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3121 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3181 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3241 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3301 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3361 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3421 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3481 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3541 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3601 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3661 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3721 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3781 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3841 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3901 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3961 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4021 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4081 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4141 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4201 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4261 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4321 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     4381 tcacgaggcc ctttcgtc
//
