LOCUS       sequence-82075-e        6181 bp    DNA     circular SYN 14-APR-2025
DEFINITION  IPTG-inducible expression of link-NGFP negative control for in vivo 
            split GFP assembly assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6181)
  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 6181)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-link-NGFP
FEATURES             Location/Qualifiers
     source          1..6181
                     /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"
     primer_bind     complement(593..611)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      607..654
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(829..834,835..851,852..857))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(884..902)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        970..1074
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1075..1143,1144..1935)
                     /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(1293..1312)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2106..2694
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2595..2614
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2848..2865
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2880..3022
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2948..2967)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3108..3130
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3124..3315)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4627..5709)
                     /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     5671..5690
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5710..5787)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5993..6012
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6096..6115
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6096..6114
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6115..6139
                     /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             6154..6176
                     /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 ctcgagcaat cccgggaatt
      541 aaggatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
      601 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
      661 ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata accaagccta
      721 tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca
      781 tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat
      841 cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta
      901 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
      961 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1021 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     1081 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     1141 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1201 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1261 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     1321 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1381 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1441 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1501 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1561 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     1621 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1681 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1741 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1801 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1861 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1921 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1981 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2041 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2101 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2161 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2221 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     2281 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2341 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2401 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2461 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2521 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2581 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2641 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2701 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2761 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2821 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2881 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca
     2941 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3001 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     3061 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3121 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     3181 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     3241 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3301 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     3361 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     3421 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     3481 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     3541 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     3601 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     3661 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     3721 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     3781 tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc
     3841 gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt
     3901 ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc
     3961 gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
     4021 gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg
     4081 acgatcagcg gtccagtgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc
     4141 tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg
     4201 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac
     4261 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
     4321 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     4381 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     4441 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4501 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4561 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     4621 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4681 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     4741 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     4801 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     4861 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     4921 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     4981 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     5041 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     5101 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     5161 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     5221 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     5281 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     5341 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     5401 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     5461 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     5521 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     5581 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     5641 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     5701 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     5761 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     5821 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     5881 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     5941 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     6001 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     6061 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
     6121 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
     6181 c
//
