LOCUS       sequence-133191-        7063 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U+36GFP-BirA for expression in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7063)
  AUTHORS   Li M, Tao Y, Shu Y, LaRochelle JR, Steinauer A, Thompson D, 
            Schepartz A, Chen ZY, Liu DR
  TITLE     Discovery and Characterization of a Peptide That Enhances Endosomal 
            Escape of Delivered Proteins in Vitro and in Vivo.
  JOURNAL   J Am Chem Soc. 2015 Nov 11;137(44):14084-93. doi: 
            10.1021/jacs.5b05694. Epub 2015 Oct 30.
  PUBMED    26465072
REFERENCE   2  (bases 1 to 7063)
  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: 
            "10.1021/jacs.5b05694"; journalName: "J Am Chem Soc"; date: 
            "2015-11-11- 11"; volume: "137"; issue: "44"; pages: "14084-93"
COMMENT     Sequence Label: U_(+36GFP)-BirA
FEATURES             Location/Qualifiers
     source          1..7063
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    2..26
                     /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             41..63
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1003..1962
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1003..1962
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1972..1989
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2008..2052
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2090..2108)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2104..2151
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2188..2643
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2275..2294)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2485..2506
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2758..3549,3550..3618))
                     /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     3381..3400
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3619..3711)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3792..4380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4281..4300
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4534..4551
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4566..4708
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4634..4653)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4794..4816
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4810..5001)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5573..6655)
                     /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     6617..6636
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6656..6733)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6939..6958
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
       61 agatatcata tgggcctgtt tgatattatt aaaaaagtgg cgagcgtgat tggcggcctg
      121 gctagcggtg gtagcggtgg ttctggtggt tctggtggta gcggcggtag cggtggtagc
      181 ggtggtagcg gtggcagcgg cggttctagc aaaggtgaac gtctgtttcg tggtaaagta
      241 ccgatcttag tggaattaaa gggcgacgtg aacggtcata aatttagcgt gcgcggcaaa
      301 ggcaaaggtg acgctacccg tggtaaattg accctgaagt ttatttgcac aacaggcaaa
      361 ttacccgttc cgtggcccac cttagtgacc accctgacct atggcgttca gtgcttcagt
      421 cgttacccta aacacatgaa acgtcacgat tttttcaaat cagccatgcc taaaggatat
      481 gttcaagagc gtacaatcag cttcaagaag gatggcaaat ataaaacgcg tgcggaagtg
      541 aaatttgaag gccgcacatt agtaaatcgt atcaaactga aaggtcgtga cttcaaagaa
      601 aaaggcaaca ttttaggcca taaactgcgt tataacttta attctcataa ggtgtatatt
      661 acggccgata aacgcaagaa tggtatcaag gcaaaattca aaattcgcca taacgtgaaa
      721 gacggcagcg ttcaattagc ggatcattat caacaaaaca cgccgattgg tcgcgggcct
      781 gtactgttac ctcgcaacca ctacctgagc acccgttcta aactgagcaa agatccgaaa
      841 gaaaaacgcg atcacatggt tctgttagaa ttcgtgaccg ctgcaggcat taagcacgga
      901 cgcgacgaac gctacaagac cggtggtagc ggtggttctg gtggttctgg tggtagcggc
      961 ggtagcggtg gtagcggtgg tagcggtggc agcggcggta ccaaggataa caccgtgcca
     1021 ctgaaattga ttgccctgtt agcgaacggt gaatttcact ctggcgagca gttgggtgaa
     1081 acgctgggaa tgagccgggc ggctattaat aaacacattc agacactgcg tgactggggc
     1141 gttgatgtct ttaccgttcc gggtaaagga tacagcctgc ctgagcctat ccagttactt
     1201 aatgctaaac agatattggg tcagctggat ggcggtagtg tagccgtgct gccagtgatt
     1261 gactccacga atcagtacct tcttgatcgt atcggagagc ttaaatcggg cgatgcttgc
     1321 attgcagaat accagcaggc tggccgtggt cgccggggtc ggaaatggtt ttcgcctttt
     1381 ggcgcaaact tatatttgtc gatgttctgg cgtctggaac aaggcccggc ggcggcgatt
     1441 ggtttaagtc tggttatcgg tatcgtgatg gcggaagtat tacgcaagct gggtgcagat
     1501 aaagttcgtg ttaaatggcc taatgacctc tatctgcagg atcgcaagct ggcaggcatt
     1561 ctggttgagc tgactggcaa aactggcgat gcggcgcaaa tagtcattgg agccgggatc
     1621 aacatggcaa tgcgccgtgt tgaagagagt gtcgttaatc aggggtggat cacgctgcag
     1681 gaagcgggga tcaatctcga tcgtaatacg ttggcggcca tgctaatacg tgaattacgt
     1741 gctgcgttgg aactcttcga acaagaagga ttggcacctt atctgtcgcg ctgggaaaag
     1801 ctggataatt ttattaatcg cccagtgaaa cttatcattg gtgataaaga aatatttggc
     1861 atttcacgcg gaatagacaa acagggggct ttattacttg agcaggatgg aataataaaa
     1921 ccctggatgg gcggtgaaat atccctgcgt agtgcagaaa aaaggggatc ccatcaccac
     1981 caccatcact aatgactcga gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag
     2041 cacatggact cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa
     2101 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     2161 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     2221 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2281 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2341 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2401 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2461 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2521 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2581 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     2641 tttctggcgg cacgatggca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     2701 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     2761 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     2821 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     2881 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     2941 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3001 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3061 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3121 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3181 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3241 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3301 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3361 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3421 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3481 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3541 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3601 gaaatgttga atactcatac tcttcctttt tcaatcatga ttgaagcatt tatcagggtt
     3661 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggtcatg
     3721 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3781 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3841 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3901 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3961 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4021 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4081 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4141 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4201 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4261 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4321 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4381 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4441 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4501 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4561 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatat
     4621 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4681 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4741 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4801 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     4861 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4921 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4981 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     5041 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     5101 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     5161 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     5221 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     5281 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     5341 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     5401 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgct
     5461 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     5521 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     5581 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5641 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     5701 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     5761 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     5821 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
     5881 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5941 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     6001 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     6061 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     6121 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     6181 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     6241 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     6301 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     6361 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     6421 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     6481 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     6541 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     6601 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     6661 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     6721 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     6781 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     6841 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     6901 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6961 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     7021 gagatcgatc tcgatcccgc gaaattaata cgactcacta tav
//
