LOCUS       sequence-359611-        5871 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tripartite split-GFP. E. coli vector. Expression of GFP1-9 detector 
            fragment..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5871)
  AUTHORS   Pedelacq JD, Waldo GS, Cabantous S
  TITLE     High-Throughput Protein-Protein Interaction Assays Using Tripartite 
            Split-GFP Complementation.
  JOURNAL   Methods Mol Biol. 2019;2025:423-437. doi: 
            10.1007/978-1-4939-9624-7_20.
  PUBMED    31267465
REFERENCE   2  (bases 1 to 5871)
  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: "Methods Mol
            Biol."; date: "2019"; volume: "2025:423-437. doi"; pages: " 
            10.1007/978-1-4939-9624-7_20"
COMMENT     Sequence Label: pET N6his GFP1-9
FEATURES             Location/Qualifiers
     source          1..5871
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..44
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        25..43
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    44..68
                     /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             83..105
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             124..141
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             151..168
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             772..789
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(842..860)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      856..903
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      940..1395
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1027..1046)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1237..1258
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1487..2302)
                     /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(1487..2302)
                     /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     2210..2229
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2294..2313
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2449..2994
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     2895..2914
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    3186..3325
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3251..3270)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3411..3433
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3427..3618)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRNCLARFGDDGENL"
     CDS             complement(4427..5509)
                     /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     5471..5490
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5510..5587)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5793..5812
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
       61 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac catgggcagc
      121 agccatcatc atcatcatca cagcagcggc ctggtgccgc gcggcagcca tatgaggaaa
      181 ggagaagaac ttttcactgg agtcgtccca attcttattg aattagatgg tgatgttaat
      241 gggcacaaat tttttgtccg tggagagggt gaaggtgatg ctacaatcgg aaaactcagc
      301 cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact
      361 ctgacctatg gtgttcaatg cttttcccgt tatccggatc acatgaaacg gcatgacttt
      421 ttcaagagtg ccatgcccga aggttatgta caggaacgca ctatatattt caaagatgac
      481 gggacctaca agacgcgtgc tgaagtcaag tttgaaggtg atacccttgt taatcgtatc
      541 gagttaaagg gtattgattt taaagaagat ggaaacattc ttggacacaa actcgagtac
      601 aactttaact cacacaaagt atacatcacg gcagacaaac aaaataatgg aatcaaagct
      661 aacttcacaa ttcgccacaa cgttgaagat ggttccgttc aactagcaga ccattatcaa
      721 caaaatactc caattggcga tggccctgtc ctttaaggat cctaactcga gcaccaccac
      781 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
      841 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      901 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
      961 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1021 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1081 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1141 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1201 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1261 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1321 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1381 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1441 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     1501 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     1561 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     1621 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     1681 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     1741 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     1801 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     1861 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     1921 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     1981 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     2041 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     2101 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     2161 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     2221 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     2281 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     2341 ttattgtaac aacttatatc gtatggggct gacttcaggt gctacatttg aagagataaa
     2401 ttgcactgaa atctagtaat attttatctg attaataaga tgatcttctt gagatcgttt
     2461 tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaact gccttgcagg gcggtttttc
     2521 gaaggttctc tgagctacca actctttgaa ccgaggtaac tggcttggag gagcgcagtc
     2581 accaaaactt gtcctttcag tttagcctta accggcgcat gacttcaaga ctaactcctc
     2641 taaatcaatt accagtggct gctgccagtg gtgcttttgc atgtctttcc gggttggact
     2701 caagacgata gttaccggat aaggcgcagc ggtcggactg aacggggggt tcgtgcatac
     2761 agtccagctt ggagcgaact gcctacccgg aactgagtgt caggcgtgga atgagacaaa
     2821 cgcggccata acagcggaat gacaccggta aaccgaaagg caggaacagg agagcgcacg
     2881 agggagccgc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac
     2941 tgatttgagc gtcagatttc gtgatgcttg tcaggggggc ggagcctatg gaaaaacggc
     3001 tttgccgcgg ccctctcact tacctgttaa gtatcttcct ggcatcttcc aggaaatctc
     3061 cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac gaccgagcgt agcgagtcag
     3121 tgagcgagga agcggaatat atccaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3181 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     3241 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     3301 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     3361 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3421 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagttgc ggtaaagctc
     3481 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     3541 gagtttctcc agaagcgtta atgtctggct tctgataaaa cgggccatgt taagggcggt
     3601 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     3661 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     3721 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     3781 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     3841 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     3901 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     3961 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgatttat tctgctaacc
     4021 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgctcac
     4081 tcgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     4141 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     4201 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     4261 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     4321 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     4381 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     4441 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4501 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     4561 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     4621 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     4681 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     4741 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     4801 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     4861 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     4921 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     4981 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     5041 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     5101 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     5161 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     5221 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     5281 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     5341 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     5401 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     5461 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     5521 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     5581 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     5641 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     5701 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     5761 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     5821 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag g
//
