LOCUS       Exported                6267 bp DNA     circular SYN 02-JUL-2021
DEFINITION  BBF10K_000528 in pOpen_v4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6267)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 2, 2021 from SnapGene 5.3.1
            https://www.snapgene.com
COMMENT     This file was created using tools provided by Twist Bioscience
FEATURES             Location/Qualifiers
     source          1..6267
                     /organism="BBF10K_000528 in pOpen_v4"
     primer_bind     21..51
                     /label=Gibson-for
     CDS             52..1773
                     /codon_start=1
                     /product="anti-CD19 chimeric antigen receptor, 
                     intracellular 2B4"
                     /label=CD19CAR-2B4
                     /translation="MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISC
                     RASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQED
                     IATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQ
                     SLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSK
                     SQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTP
                     APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCK
                     WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTS
                     QEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDV
                     YSCKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
                     EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR"
     primer_bind     complement(1774..1798)
                     /label=Gibson-rev
     terminator      1917..1964
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1969..1985)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2144..2394
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(2397..3152)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      3243..3462
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     protein_bind    3882..3903
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3918..3948
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3956..3972
                     /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     3980..3996
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4069..4914
                     /codon_start=1
                     /label=repL
                     /translation="MLAKVTFLSCITMSDFTFSGYELACFVTHSGLSRSAGHILSQCAN
                     LAATTSEYFIHKPHRLIAAETGYSQSTVVRAFREAVNKGILSVEIVIGDHRERRANLYR
                     FTPSFLAFAQQAKNALIESKLKISSAATKVKAVLAKTLALFNFLSTPPCQNDTPSPCQD
                     DVAIKNKKSQVKKTKRSVSGGAGTTSLKKLTSWIAKAKAKADNLRLSKKRTQKHEFKQK
                     VEAAARKYAYLKNKRSPDIGGISNFDNLPHCMTVNEALNAVLAKNKDNEQWGIPAGFRG
                     "
     misc_feature    5022..5173
                     /label=nnpp promoters
     CDS             5213..6028
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      complement(6138..6181)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     6199..6215
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      6216..6247
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 aagaattact gacccctcgg cagactgagt cgcccggggg gatccgccac catgctgctg
       61 ctggtcactt ccctgctctt gtgtgaactc ccgcatcctg cttttctcct cattcctgat
      121 attcaaatga cgcaaactac aagttccctc tctgcttccc ttggagaccg cgtcacaatt
      181 agttgtcgcg catcacaaga tatttctaaa tatttgaatt ggtaccaaca aaagccagat
      241 ggaactgtaa aactcctcat ttatcatacg agtcggctcc attctggagt tccttctagg
      301 ttctccggat caggatcagg aactgattat tctctcacta taagcaatct tgagcaagag
      361 gacatagcga cttacttttg ccaacagggg aatacgctcc cttatacttt tggcggcggg
      421 acgaaactcg agattacagg gagtacttca ggttccggga aaccaggtag cggtgaaggt
      481 agtactaaag gggaagtcaa gctccaagag tccggtcccg gcttggtcgc gccgtcacaa
      541 agtctctccg ttacatgcac tgtctctggt gtatctttgc cggattacgg agtcagctgg
      601 attaggcaac cgcctcggaa agggctcgag tggctcgggg tcatttgggg atcagaaacc
      661 acgtattata attccgcact caaatcaaga ctcacgatca ttaaagacaa tagcaagtcc
      721 caagttttcc tgaagatgaa ttcactgcaa acagatgaca ctgcgattta ttattgtgcg
      781 aaacattact actatggtgg gtcctatgca atggattatt ggggacaagg aacatccgtt
      841 acagtcagct ccgctgcagc cacaacaact cctgccccca ggccccctac cccagctcct
      901 acaattgcat cacaacctct ctctttgcgt cctgaagcct gtagacccgc cgctggcgga
      961 gccgtccata caagaggtct cgattttgct tgcgacattt atatatgggc tccactggct
     1021 ggcacatgcg gtgttctcct gttgagcttg gtaattacac tgtattgtaa gtggagacgc
     1081 aaacgtaaag aaaagcaaag tgaaacgtct ccgaaagagt tcctgacgat ctatgaggac
     1141 gttaaagact tgaagactcg tcggaaccat gaacaagaac aaaccttccc cggtggtggt
     1201 tctacaattt attccatgat acaaagccaa agtagcgccc ctacatccca ggagccagcc
     1261 tacaccctct acagtctgat acaacccagc agaaaatccg gctcacgtaa acgcaatcat
     1321 tccccctcat ttaactccac catttacgag gttataggta aatcccagcc caaggcacaa
     1381 aatccagcac ggctgagtag aaaggaactt gaaaatttcg acgtatactc ttgcaaacgc
     1441 gttaaatttt cccggtccgc tgatgcaccg gcatatcaac aaggacaaaa ccaattgtat
     1501 aatgaattga atttgggacg tcgcgaggaa tatgatgtgc ttgacaaaag gcggggaaga
     1561 gatccggaaa tgggtggaaa acccagacgc aagaatccgc aagagggtct ctacaatgag
     1621 ctccaaaagg ataaaatggc agaagcttat tctgaaattg ggatgaaagg ggaaagaaga
     1681 cgtggtaaag gacatgatgg tttgtaccaa gggcttagca cagcgactaa ggatacctac
     1741 gacgccctgc acatgcaggc cctgccccca aggggatccg ccaccaactt cagcctgcta
     1801 gtaaagcata gtgcccagct agctttctga ctattatgag tagggataac agggtaatat
     1861 gtagtccagg gggaaatggc gctgaggtgt caatcgtcgg agccgctgag caataactag
     1921 cataacccct tggggcctct aaacgggtct tgaggggttt tttgcatggt catagctgtt
     1981 tcctgagagc ttggcaggtg tcgcgtatgc atcgatcacg tgtccacgtt ctttaatagt
     2041 ggactcttgt tccaaactgg aacaacactc ggatcgatcc ggcgcgcacc gtgggaaaaa
     2101 ctccaggtag aggtacacac gcggatagcc aattcagagt aataaactgt gataatcaac
     2161 cctcatcaat gatgacgaac taacccccga tatcaggtca catgacgaag ggaaagagaa
     2221 ggaaatcaac tgtgacaaac tgccctcaaa tttggcttcc ttaaaaatta cagttcaaaa
     2281 agtatgagaa aatccatgca ggctgaagga aacagcaaaa ctgtgacaaa ttaccctcag
     2341 taggtcagaa caaatgtgac gaaccaccct caaatctgtg acagataacc ctcagactat
     2401 cctgtcgtca tggaagtgat atcgcggaag gaaaatacga tatgagtcgt ctggcggcct
     2461 ttctttttct caatgtatga gaggcgcatt ggagttctgc tgttgatctc attaacacag
     2521 acttgcagga agcggcggcg gaagtcaggc atacgctggt aactttgagg cagctggtaa
     2581 cgctctatga tccagtcgat tttcagagac acgatgcctg agccatccgg cttacgatac
     2641 tgacacaggg attcgtataa acgcatggca tacggattgg tgatttcttt tgtttcacta
     2701 agccgaaact gcgtaaaccg gttctgtaac ccgataaaga agggaatgag atatgggttg
     2761 atatgtacac tgtaaagccc tctggatgga ctgtgcgcac gtttgataaa ccaaggaaaa
     2821 gattcatagc ctttttcatc accggcatcc tcttcagggc gataaaaaac cacttccttc
     2881 cccgcgaaac tcttcaatgc ctgccgtata tccttactgg cttccgcaga ggtcaatccg
     2941 aatatttcag catatttagc aacatggatc tcgcagatac cgtcatgttc ctgtagggtg
     3001 ccatcagatt ttctgatctg gtcaacgaac agatacagca tacgtttttg atcccgggag
     3061 agactatatg ccgcctcagt gaggtcgttt gactggacga ttcgcgggct atttttacgt
     3121 ttcttgtgat tgataaccgc tgtttccgcc atgacagatc catgtgaagt gtgacaagtt
     3181 tttagattgt cacactaaat aaaaaagagt caataagcag ggataacttt gtgaaaaaac
     3241 agcttcttct gagggcaatt tgtcacaggg ttaagggcaa tttgtcacag acaggactgt
     3301 catttgaggg tgatttgtca cactgaaagg gcaatttgtc acaacacctt ctgtagaacc
     3361 agcatggata aaggcctaca aggcgctcta aaaaagaaga tctaaaaact ataaaaaaaa
     3421 taattataaa aatatccccg tggataagtg gataacccca agggaagttt tttcaggcat
     3481 cgtgtgtaag cagaatatat aagtgctgtt ccctggtgct tcctcgctca ctcgaaattc
     3541 ccggggatag ctttatgctt gtaaaccgtt ttgtgaaaaa atttttaaaa taaaaaaggg
     3601 gacctctagg gtccccaatt aattagtaat ataatctatt aaaggtcatt caaaaggtca
     3661 tccaccggat caattcccct gctcgcgcag gctgggtgcc aagctctcgg gtaacatcaa
     3721 ggcccgatcc ttggagccct tgccctcccg cacgatgatc gtgccgtgat cgaaatccag
     3781 atccttgacc cgcagttgca aaccctcact gatccgattc attaatgcag ctggcacgac
     3841 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     3901 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     3961 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcggtacc
     4021 tgtcgcggca acgcgctaac agacgtagta agaaccacca gcattgtaat gctggctaaa
     4081 gtcactttcc tgagctgtat aacgatgagc gattttactt tttctggcta tgaattggcc
     4141 tgctttgtaa cacactccgg tctatcccgt agcgccgggc atatcctgtc gcaatgtgca
     4201 aatctcgcgg caacaaccag tgaatacttc attcacaagc ctcaccgcct gatcgcggca
     4261 gaaactggtt atagccaatc aaccgtcgtt cgtgcattcc gtgaagctgt aaacaaaggt
     4321 atcctgtctg tagagattgt tatcggcgat caccgtgaac gtcgcgctaa cctgtaccgg
     4381 tttacaccat cctttttggc cttcgcacaa caagccaaaa atgcgctgat agaaagcaaa
     4441 ttaaagatct cttcagcggc aaccaaggtt aaagctgttc tcgctaagac attggcttta
     4501 tttaattttt tatccacacc cccatgtcaa aatgataccc cctccccctg tcaggatgac
     4561 gtggcaataa agaataagaa gtcacaagtt aaaaaaacaa aaagatcagt ttccggcggt
     4621 gccggaacaa ccagcctcaa aaaattgact tcatggatcg ctaaggcaaa agcaaaggct
     4681 gacaatctgc ggttatccaa aaaacgcact caaaaacatg agttcaagca gaaagtagag
     4741 gcggctgcgc ggaaatatgc ttacctgaag aacaagcgtt cgcctgatat tggcgggata
     4801 tcaaacttcg ataacctacc gcattgcatg acggtaaacg aagctcttaa tgcggtttta
     4861 gccaaaaata aagataacga acaatggggt ataccggcag gattcagagg gtaatgaatt
     4921 gctctaatta taaccatgca tactttcaac acctctagtt tgccatgagg caaactcata
     4981 ggtgtcctgg taagaggaca ctgttgccaa aactggacgc cccattattg caattaataa
     5041 acaactaacg gacaattcta cctaacaata agtggagttg cggccggccg cgatccgcta
     5101 gcaaagccac gttgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     5161 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     5221 tattcaacgg gaaacgtctt gctcgcgccc gcgattaaat tccaacatgg atgctgattt
     5281 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt
     5341 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     5401 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     5461 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     5521 gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     5581 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     5641 cgatcgcgta tttcgcctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     5701 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     5761 taaactgttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     5821 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     5881 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     5941 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     6001 tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggcg
     6061 agctctgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     6121 gcgtatcacg aggcagaatt tcagataaaa aaaatcctta gctttcgcta aggatgattt
     6181 ctgcacctgc accagtcagt aaaacgacgg ccagtagtca aaagcctccg accggaggct
     6241 tttgacttgg ttcaggtgga gtgggag
//
