LOCUS       sequence-82261-e        6256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG-inducible expression of Z-NGFP positive control for in vivo 
            split GFP assembly assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6256)
  AUTHORS   Magliery TJ, Wilson CG, Pan W, Mishler D, Ghosh I, Hamilton AD, 
            Regan L
  TITLE     Detecting protein-protein interactions with a green fluorescent 
            protein fragment reassembly trap: scope and mechanism.
  JOURNAL   J Am Chem Soc. 2005 Jan 12;127(1):146-57.
  PUBMED    15631464
REFERENCE   2  (bases 1 to 6256)
  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: "J Am Chem 
            Soc."; date: "2005-01-12"; volume: "127(1)"; pages: "146-57"
COMMENT     Sequence Label: pET11a-Z-NGFP
FEATURES             Location/Qualifiers
     source          1..6256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             12..29
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(63..91)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             579..590
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(668..686)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      682..729
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(904..909,910..926,927..932))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(959..977)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1045..1149
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1150..1218,1219..2010)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1368..1387)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2181..2769
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2670..2689
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2923..2940
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2955..3097
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3023..3042)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3183..3205
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3199..3390)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4702..5784)
                     /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     5746..5765
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5785..5862)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6068..6087
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6171..6190
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6171..6189
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6190..6214
                     /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             6229..6251
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atatggctag ccatcaccac catcaccatg gcgcgagcaa aggagaagaa ctcttcactg
       61 gagttgtccc aattcttgtt gaattagatg gtgatgttaa cggccacaag ttctctgtca
      121 gtggagaggg tgaaggtgat gcaacatacg gaaaacttac cctgaagttc atctgcacta
      181 ctggcaaact gcctgttcca tggccaacac tagtcactac tctgtgctat ggtgttcaat
      241 gcttttcaag atacccggat catatgaaac ggcatgactt tttcaagagt gccatgcccg
      301 aaggttatgt acaggaaagg accatcttct tcaaagatga cggcaactac aagacacgtg
      361 ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgact
      421 tcaaggaaga tggcaacatt ctgggacaca aattggaata caactataac tcacacaacg
      481 ttctcatcat ggcagacaaa caaggtggct ctggctctgg ctcgagcgcc ctcaaaaaag
      541 aattgcaggc aaacaaaaaa gaacttgcgc agctgaagtg ggagttacaa gctctgaaaa
      601 aggaactggc gcagtaagga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      661 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      721 ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg
      781 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
      841 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
      901 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
      961 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     1021 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1081 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1141 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1201 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1261 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1321 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1381 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1441 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1501 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1561 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1621 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     1681 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     1741 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     1801 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     1861 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     1921 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     1981 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2041 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2101 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2161 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2221 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2281 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2341 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2401 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2461 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2521 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2581 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2641 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2701 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2761 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2821 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     2881 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     2941 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3001 cgcatatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3061 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3121 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3181 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3241 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3301 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     3361 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     3421 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     3481 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     3541 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     3601 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     3661 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     3721 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     3781 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     3841 gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct
     3901 ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct
     3961 ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg
     4021 gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag
     4081 acaaggtata gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg
     4141 gcataaatcg ccgtgacgat cagcggtcca gtgatcgaag ttaggctggt aagagccgcg
     4201 agcgatcctt gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc
     4261 aacgcgggca tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag
     4321 cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata
     4381 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4441 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4501 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4561 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4621 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4681 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4741 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4801 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4861 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4921 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4981 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5041 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5101 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5161 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5221 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5281 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5341 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5401 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5461 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5521 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5581 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5641 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5701 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5761 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5821 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5881 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     5941 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6001 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6061 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6121 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     6181 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     6241 taagaaggag atatac
//
