LOCUS       sequence-176149-        5893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP with +16 theoretical charge in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5893)
  AUTHORS   Thompson DB, Villasenor R, Dorr BM, Zerial M, Liu DR
  TITLE     Cellular uptake mechanisms and endosomal trafficking of supercharged
            proteins.
  JOURNAL   Chem Biol. 2012 Jul 27;19(7):831-43. doi: 
            10.1016/j.chembiol.2012.06.014.
  PUBMED    22840771
REFERENCE   2  (bases 1 to 5893)
  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: 
            "10.1016/j.chembiol.2012.06"; journalName: "Chem Biol"; date: 
            "2012-07-27- 27"; volume: "19"; issue: "7"; pages: "831-43"
COMMENT     Sequence Label: pET-6xHis-(pos16)GFP
FEATURES             Location/Qualifiers
     source          1..5893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1234..1425
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1419..1441)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1527..1666
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1582..1601
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1681..1698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1852..2440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1932..1951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2521..2613
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2614..2682,2683..3474)
                     /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     complement(2832..2851)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3589..4044)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3726..3747)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3938..3957
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4081..4128)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4124..4142
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4180..4224)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(4972..4989)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5002..5024
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5039..5063
                     /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)."
     primer_bind     complement(5063..5082)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5064..5082)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5170..5189)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5395..5472
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5473..5893,1..662)
                     /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     complement(5492..5511)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt
       61 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
      121 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
      181 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
      241 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      301 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      361 ggctgcgcgt tggtgcggac atctcggtag tgggatacga cgataccgaa gacagctcat
      421 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      481 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      541 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      601 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      661 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
      721 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactagcatg
      781 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
      841 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
      901 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
      961 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt
     1021 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg
     1081 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt
     1141 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc
     1201 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt
     1261 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg
     1321 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac
     1381 cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     1441 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     1501 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     1561 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg
     1621 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
     1681 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1741 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     1801 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     1861 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     1921 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     1981 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2041 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2101 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2161 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2221 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2281 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2341 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2401 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2461 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaccta
     2521 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2581 aaatgcttca atcatgattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2641 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2701 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2761 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2821 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2881 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2941 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     3001 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3061 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3121 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     3181 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3241 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3301 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3361 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3421 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3481 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3541 atctaggtga agatcctttt tgataatctc atgccatcgt gccgccagaa attgtaaacg
     3601 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     3661 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     3721 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     3781 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     3841 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     3901 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     3961 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     4021 ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg gatatagttc ctcctttcag
     4081 caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt attgctcagc
     4141 ggtggcagca gcctaggtta attaagctgc gctagtagac gagtccatgt gctggcgttc
     4201 aaatttcgca gcagcggttt ctttaccaga ctcgagggta cctcattatt acttgtagag
     4261 ttcgtccatt ccgtgagtaa tgcctgcagc ggtcacgaat tctaacagaa ccatgtgatc
     4321 gcgtttttct ttcggatctt tgctcagtgc tgaacgggtg ctcaggtagt ggttgcgagg
     4381 taacagtaca ggcccgcgac caatcggcgt gttttgttga taatgatccg ctaattgaac
     4441 gctgccgtct ttcacgttat ggcgaatttt gaaatttgcc ttgataccat tcttgcgttt
     4501 atcggccgta atatacacct tatgagaatt aaagttataa cgcagcttat ggcctaaaat
     4561 gttgcctttt tctttgaagt cacgaccttt cagtttgata cgatttacta atgtgcggcc
     4621 tttaaatttc acttccgcac gcgttttata tttgccatcc ttcttgaagc tgattgtacg
     4681 ctcttgaaca tatcctttag gcatggctga tttgaaaaaa tcgtgacgtt tcatatgttt
     4741 agggtaacga ctgaagcact gaacgccata ggtcagggtg gtcactaagg tgggccacgg
     4801 aacgggtaat ttgcctgttg tgcaaataaa cttcagggtc aatttaccac gggtagcgtc
     4861 accttcgcct tcgccgcgca cgctaaattt gtgaccgttc acgtcgccgt ctaattccac
     4921 taagatcggt actacacccg taaacagacg ttcacctttg ctagcgccac cgtgatggtg
     4981 gtggtgatga cccatggtat atctccttct taaagttaaa caaaattatt tctagagggg
     5041 aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat
     5101 cgatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
     5161 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
     5221 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
     5281 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     5341 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
     5401 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     5461 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     5521 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     5581 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     5641 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     5701 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     5761 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     5821 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     5881 gaagctgcct gca
//
