pTXB1-APEX2-F

LOCUS       pTXB1-APEX2-F                7428 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression of C-terminally FLAG-tagged APEX2 peroxidase.
ACCESSION   .
VERSION     .
KEYWORDS    pTXB1-APEX2-F
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7428)
  TITLE     Dual DNA/protein tagging of open chromatin unveils dynamics of 
            epigenomic landscapes in leukemia
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7428)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..7428
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     126..148
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(142..333)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(890..1972)
                     /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     1934..1953
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1973..2050)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      2203..2289
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2386..2472
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2569..2655
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2752..2838
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2935..3021
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3156..3175
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3156..3174
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3175..3199
                     /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)."
     CDS             3243..3992
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="MGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAG
                     TFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVA
                     VEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGH
                     TIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVD
                     KYAADEDAFFADYAEAHQKLSELGFADA"
     CDS             3993..4016
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4017..4610
                     /codon_start=1
                     /gene="Mycobacterium xenopi gyrA"
                     /product="modified GyrA intein (Southworth et al., 1999)"
                     /label=Mxe GyrA intein
                     /note="N-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
                     LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
                     IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
                     AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
     CDS             4641..4796
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     primer_bind     complement(4861..4879)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4875..4922
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4982..5086
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5087..5947
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5305..5324)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5989..6502)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6184..6205)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6396..6415
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      6613..7201
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7102..7121
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7355..7372
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(7394..7413)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
       61 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
      121 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
      181 gctgcggtaa agctcatcag cgtggtcgtg cagcgattca cagatgtctg cctgttcatc
      241 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
      301 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
      361 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
      421 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
      481 gcgggaccag agaaaaatca ctcagggtca atgccagccg aacgccagca agacgtagcc
      541 cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
      601 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
      661 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
      721 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
      781 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
      841 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
      901 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
      961 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     1021 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     1081 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     1141 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     1201 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     1261 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     1321 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     1381 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     1441 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     1501 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     1561 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     1621 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     1681 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     1741 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     1801 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     1861 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     1921 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     1981 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     2041 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     2101 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg ccggcatgcc
     2161 gccctttcgt cttcaagaat taattcccaa ttccccaggc atcaaataaa acgaaaggct
     2221 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     2281 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     2341 gcaggacgcc cgccataaac tgccaggaat taattcccca ggcatcaaat aaaacgaaag
     2401 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     2461 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     2521 cgggcaggac gcccgccata aactgccagg aattaattcc ccaggcatca aataaaacga
     2581 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     2641 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     2701 tggcgggcag gacgcccgcc ataaactgcc aggaattaat tccccaggca tcaaataaaa
     2761 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     2821 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     2881 gggtggcggg caggacgccc gccataaact gccaggaatt aattccccag gcatcaaata
     2941 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     3001 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
     3061 ggagggtggc gggcaggacg cccgccataa actgccagga attggggatc ggaattaatt
     3121 cccggtttaa accggggatc tcgatcccgc gaaattaata cgactcacta taggggaatt
     3181 gtgagcggat aacaattccc ctctagaaat aattttgtta actttaagaa ggagatatac
     3241 atatgggaaa gtcttaccca actgtgagtg ctgattacca ggacgccgtt gagaaggcga
     3301 agaagaagct cagaggcttc atcgctgaga agagatgcgc tcctctaatg ctccgtttgg
     3361 cattccactc tgctggaacc tttgacaagg gcacgaagac cggtggaccc ttcggaacca
     3421 tcaagcaccc tgccgaactg gctcacagcg ctaacaacgg tcttgacatc gctgttaggc
     3481 ttttggagcc actcaaggcg gagttcccta ttttgagcta cgccgatttc taccagttgg
     3541 ctggcgttgt tgccgttgag gtcacgggtg gacctaaggt tccattccac cctggaagag
     3601 aggacaagcc tgagccacca ccagagggtc gcttgcccga tcccactaag ggttctgacc
     3661 atttgagaga tgtgtttggc aaagctatgg ggcttactga ccaagatatc gttgctctat
     3721 ctgggggtca cactattgga gctgcacaca aggagcgttc tggatttgag ggtccctgga
     3781 cctctaatcc tcttattttc gacaactcat acttcacgga gttgttgagt ggtgagaagg
     3841 aaggtctcct tcagctacct tctgacaagg ctcttttgtc tgaccctgta ttccgccctc
     3901 tcgttgacaa atatgcagcg gacgaagatg ccttctttgc tgattacgct gaggctcacc
     3961 aaaagctttc cgagcttggg tttgctgatg ccgattacaa ggatgacgac gataagtgca
     4021 tcacgggaga tgcactagtt gccctacccg agggcgagtc ggtacgcatc gccgacatcg
     4081 tgccgggtgc gcggcccaac agtgacaacg ccatcgacct gaaagtcctt gaccggcatg
     4141 gcaatcccgt gctcgccgac cggctgttcc actccggcga gcatccggtg tacacggtgc
     4201 gtacggtcga aggtctgcgt gtgacgggca ccgcgaacca cccgttgttg tgtttggtcg
     4261 acgtcgccgg ggtgccgacc ctgctgtgga agctgatcga cgaaatcaag ccgggcgatt
     4321 acgcggtgat tcaacgcagc gcattcagcg tcgactgtgc aggttttgcc cgcgggaaac
     4381 ccgaatttgc gcccacaacc tacacagtcg gcgtccctgg actggtgcgt ttcttggaag
     4441 cacaccaccg agacccggac gcccaagcta tcgccgacga gctgaccgac gggcggttct
     4501 actacgcgaa agtcgccagt gtcaccgacg ccggcgtgca gccggtgtat agccttcgtg
     4561 tcgacacggc agaccacgcg tttatcacga acgggttcgt cagccacgct actggcctca
     4621 ccggtctgaa ctcaggcctc acgacaaatc ctggtgtatc cgcttggcag gtcaacacag
     4681 cttatactgc gggacaattg gtcacatata acggcaagac gtataaatgt ttgcagcccc
     4741 acacctcctt ggcaggatgg gaaccatcca acgttcctgc cttgtggcag cttcaatgac
     4801 tgcaggaagg ggatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     4861 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     4921 tgctgaaagg aggaactata tccggataac tacgtcaggt ggcacttttc ggggaaatgt
     4981 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5041 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5101 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5161 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5221 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgtttccc
     5281 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     5341 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5401 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5461 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5521 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5581 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5641 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5701 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5761 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5821 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5881 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5941 ttggtaactg tcagaccaag tttactcata tatactttag attgatttac cccggttgat
     6001 aatcagaaaa gccccaaaaa caggaagatt gtataagcaa atatttaaat tgtaaacgtt
     6061 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     6121 gccgaaatcg gcaaaatccc ttataaatca aaagaatagc ccgagatagg gttgagtgtt
     6181 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     6241 aaaaccgtct atcagggcga tggcccacta cgtgaaccat cacccaaatc aagttttttg
     6301 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6361 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     6421 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     6481 aatgcgccgc tatagggcgc gtaaaaggat ctaggtgaag atcctttttg ataatctcat
     6541 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6601 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6661 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6721 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     6781 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6841 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6901 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6961 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7021 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7081 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7141 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7201 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7261 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7321 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagctat
     7381 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagt
//