LOCUS       sequence-176158-        5893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP with +14 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-(pos14)GFP
FEATURES             Location/Qualifiers
     source          1..5893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1159..1350
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1344..1366)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1452..1591
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1507..1526
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1606..1623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1777..2365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1857..1876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2446..2538
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2539..2607,2608..3399)
                     /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(2757..2776)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3514..3969)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3651..3672)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3863..3882
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4006..4053)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4049..4067
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4105..4149)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(4897..4914)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4927..4949
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4964..4988
                     /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(4988..5007)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4989..5007)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5095..5114)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5320..5397
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5398..5893,1..587)
                     /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(5417..5436)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
       61 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
      121 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
      181 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
      241 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
      301 cggacatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
      361 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
      421 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
      481 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
      541 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
      601 aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc
      661 aacccagtca gctccttccg gtgggcgcgg ggcatgacta gcatgatcgt gctcctgtcg
      721 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
      781 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
      841 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
      901 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
      961 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     1021 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     1081 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     1141 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     1201 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     1261 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg
     1321 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     1381 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     1441 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     1501 actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca
     1561 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     1621 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1681 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1741 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1801 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1861 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1921 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1981 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2041 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2101 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2161 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     2221 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2281 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2341 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2401 tcagtggaac gaaaactcac gttaagggat tttggtcatg acctatttgt ttatttttct
     2461 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaatcat
     2521 gattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2581 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2641 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2701 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2761 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2821 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2881 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2941 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3001 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3061 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3121 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3181 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3241 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3301 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3361 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3421 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3481 ctttttgata atctcatgcc atcgtgccgc cagaaattgt aaacgttaat attttgttaa
     3541 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     3601 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     3661 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     3721 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     3781 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     3841 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     3901 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     3961 agggcgcgtc ccattcgcca atccggatat agttcctcct ttcagcaaaa aacccctcaa
     4021 gacccgttta gaggccccaa ggggttatgc tagttattgc tcagcggtgg cagcagccta
     4081 ggttaattaa gctgcgctag tagacgagtc catgtgctgg cgttcaaatt tcgcagcagc
     4141 ggtttcttta ccagactcga gggtacctca ttattacttg tagagttcgt ccattccgtg
     4201 agtaatgcct gcagcggtca cgaattctaa cagaaccatg tgatcgcgtt tttcgttcgg
     4261 atctttgctc agtgctgact gggtgctcag gtagtggttg tcaggtaaca gtacaggccc
     4321 gtcaccaatc ggcgtgtttt gttgataatg atccgctaat tgaacgctgc cgtcttccac
     4381 gttatggcga attttgaaat ttgccttgat accgtttttc tgtttatcgg ccgtaatata
     4441 caccttatga gaattaaagt tataacgcag cttatggcct aaaatgttgc ctttttcttt
     4501 gaagtcacga cctttcagtt tgatacgatt tactaatgtg cggcctttaa atttcacttc
     4561 cgcacgcgtt ttatatttgc catccttctt gaagctgatt gtacgctctt gaacatatcc
     4621 tttaggcatg gctgatttga aaaaatcgtg acgtttcata tgtttagggt aacgactgaa
     4681 gcactgaacg ccataggtca gggtggtcac taaggtgggc cacggaacgg gtaatttgcc
     4741 tgttgtgcaa ataaacttca gggtcaattt accacgggta gcgtcacctt cgccttcgcc
     4801 gcgcacgcta aatttgtgac cgttcacgtc gccgtctaat tccactaaga tcggtactac
     4861 acccgtaaac agacgttcac ctttgctagc gccaccgtga tggtggtggt gatgacccat
     4921 ggtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc
     4981 acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatcgatc tcgatcctct
     5041 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
     5101 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
     5161 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
     5221 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
     5281 taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa
     5341 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     5401 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     5461 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     5521 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     5581 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     5641 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     5701 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     5761 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     5821 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     5881 tcccatgaag acg
//
