LOCUS       Exported                6024 bp DNA     circular SYN 02-JUL-2021
DEFINITION  BBF10K_000529 in pOpen_v4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6024)
  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..6024
                     /organism="BBF10K_000529 in pOpen_v4"
     primer_bind     21..51
                     /label=Gibson-for
     CDS             52..1530
                     /codon_start=1
                     /product="anti-CD19 chimeric antigen receptor, 
                     intracellular 4-1BBz"
                     /label=CD19CAR-41BBz
                     /translation="MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISC
                     RASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQED
                     IATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQ
                     SLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSK
                     SQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTP
                     APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCK
                     RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQN
                     QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK
                     GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR"
     primer_bind     complement(1531..1555)
                     /label=Gibson-rev
     terminator      1674..1721
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1726..1742)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1901..2151
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(2154..2909)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      3000..3219
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     protein_bind    3639..3660
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3675..3705
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3713..3729
                     /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     3737..3753
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3826..4671
                     /codon_start=1
                     /label=repL
                     /translation="MLAKVTFLSCITMSDFTFSGYELACFVTHSGLSRSAGHILSQCAN
                     LAATTSEYFIHKPHRLIAAETGYSQSTVVRAFREAVNKGILSVEIVIGDHRERRANLYR
                     FTPSFLAFAQQAKNALIESKLKISSAATKVKAVLAKTLALFNFLSTPPCQNDTPSPCQD
                     DVAIKNKKSQVKKTKRSVSGGAGTTSLKKLTSWIAKAKAKADNLRLSKKRTQKHEFKQK
                     VEAAARKYAYLKNKRSPDIGGISNFDNLPHCMTVNEALNAVLAKNKDNEQWGIPAGFRG
                     "
     misc_feature    4779..4930
                     /label=nnpp promoters
     CDS             4970..5785
                     /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(5895..5938)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     5956..5972
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      5973..6004
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 aagaattact gacccctcgg cagactgagt cgcccggggg gatccgccac catgctgctg
       61 ttggtcactt ccctcttgtt gtgtgaactt ccacatcctg cattccttct cataccagat
      121 attcaaatga ctcaaacaac atccagtttg tccgcatctc tcggggaccg ggtaactatt
      181 tcatgtcgtg cttctcaaga tatttcaaaa tatctcaatt ggtaccaaca aaagccagat
      241 gggacagtta aactcttgat ttatcatacg tccaggctcc atagtggggt cccatccagg
      301 ttcagcggtt ctggaagtgg tacggattat tcattgacaa tttcaaattt ggagcaagag
      361 gacatagcaa cttacttttg ccaacagggg aatactctcc catatacttt tggcggtgga
      421 acaaaactcg agataacggg aagtaccagt ggaagtggga aacccgggag tggagaaggg
      481 tctacaaaag gcgaggtcaa gttgcaagag tcaggacccg gccttgttgc tccgagtcaa
      541 agcctttccg tcacatgcac tgtttctgga gtatctttgc ccgattacgg agttagttgg
      601 attcgacaac caccacgaaa aggacttgag tggcttggtg ttatatgggg ttccgaaacc
      661 acatattata actcagcgct taagagtcgt ttgacaatta ttaaagataa ttccaagtcc
      721 caagtgttcc tcaaaatgaa ttccctgcag acagacgata cagcaattta ctattgtgcg
      781 aaacattatt attatggcgg gagttatgcc atggattatt ggggacaagg tacgtctgtt
      841 actgtttctt ccgccgcagc cactactacc cccgctccta ggcccccgac tcctgccccg
      901 actattgcta gccaaccact ctctctcagg cctgaagcat gtaggcctgc tgccggcgga
      961 gccgtacata ctcgcggtct cgattttgct tgcgacatat atatttgggc acctcttgcg
     1021 ggaacatgcg gagtgctgct gctcagcctc gtaattacac tctattgtaa gagagggcgg
     1081 aagaagctgc tctacatttt caagcagcct ttcatgcgcc cggtgcagac aacacaggaa
     1141 gaggacgggt gctcttgtcg cttccccgag gaagaggaag gtggttgcga gcttcgggtc
     1201 aaattttcac gctccgccga tgctcctgca tataaacaag gacaaaatca actgtacaat
     1261 gaattgaacc tcgggcggag agaagaatat gacgtgcttg ataaacggag aggtagagat
     1321 ccagaaatgg gcggtaaacc ccggcgcaag aatccccaag agggactcta taacgagctc
     1381 caaaaggaca aaatggctga agcttattct gaaatcggaa tgaagggaga aaggagacgg
     1441 ggaaaaggcc atgacgggct gtatcaaggg ctgtccaccg cgacaaaaga tacatatgat
     1501 gcactgcata tgcaagcact gccccctcgc ggatccgcca ccaacttcag cctgctagta
     1561 gtatccgaat cgctgctgtt gttagtaagg tcgaaggtag ggataacagg gtaatgaaag
     1621 tcccaaaagc gatatgcgct gaggtgtcaa tcgtcggagc cgctgagcaa taactagcat
     1681 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gcatggtcat agctgtttcc
     1741 tgagagcttg gcaggtgtcg cgtatgcatc gatcacgtgt ccacgttctt taatagtgga
     1801 ctcttgttcc aaactggaac aacactcgga tcgatccggc gcgcaccgtg ggaaaaactc
     1861 caggtagagg tacacacgcg gatagccaat tcagagtaat aaactgtgat aatcaaccct
     1921 catcaatgat gacgaactaa cccccgatat caggtcacat gacgaaggga aagagaagga
     1981 aatcaactgt gacaaactgc cctcaaattt ggcttcctta aaaattacag ttcaaaaagt
     2041 atgagaaaat ccatgcaggc tgaaggaaac agcaaaactg tgacaaatta ccctcagtag
     2101 gtcagaacaa atgtgacgaa ccaccctcaa atctgtgaca gataaccctc agactatcct
     2161 gtcgtcatgg aagtgatatc gcggaaggaa aatacgatat gagtcgtctg gcggcctttc
     2221 tttttctcaa tgtatgagag gcgcattgga gttctgctgt tgatctcatt aacacagact
     2281 tgcaggaagc ggcggcggaa gtcaggcata cgctggtaac tttgaggcag ctggtaacgc
     2341 tctatgatcc agtcgatttt cagagacacg atgcctgagc catccggctt acgatactga
     2401 cacagggatt cgtataaacg catggcatac ggattggtga tttcttttgt ttcactaagc
     2461 cgaaactgcg taaaccggtt ctgtaacccg ataaagaagg gaatgagata tgggttgata
     2521 tgtacactgt aaagccctct ggatggactg tgcgcacgtt tgataaacca aggaaaagat
     2581 tcatagcctt tttcatcacc ggcatcctct tcagggcgat aaaaaaccac ttccttcccc
     2641 gcgaaactct tcaatgcctg ccgtatatcc ttactggctt ccgcagaggt caatccgaat
     2701 atttcagcat atttagcaac atggatctcg cagataccgt catgttcctg tagggtgcca
     2761 tcagattttc tgatctggtc aacgaacaga tacagcatac gtttttgatc ccgggagaga
     2821 ctatatgccg cctcagtgag gtcgtttgac tggacgattc gcgggctatt tttacgtttc
     2881 ttgtgattga taaccgctgt ttccgccatg acagatccat gtgaagtgtg acaagttttt
     2941 agattgtcac actaaataaa aaagagtcaa taagcaggga taactttgtg aaaaaacagc
     3001 ttcttctgag ggcaatttgt cacagggtta agggcaattt gtcacagaca ggactgtcat
     3061 ttgagggtga tttgtcacac tgaaagggca atttgtcaca acaccttctg tagaaccagc
     3121 atggataaag gcctacaagg cgctctaaaa aagaagatct aaaaactata aaaaaaataa
     3181 ttataaaaat atccccgtgg ataagtggat aaccccaagg gaagtttttt caggcatcgt
     3241 gtgtaagcag aatatataag tgctgttccc tggtgcttcc tcgctcactc gaaattcccg
     3301 gggatagctt tatgcttgta aaccgttttg tgaaaaaatt tttaaaataa aaaaggggac
     3361 ctctagggtc cccaattaat tagtaatata atctattaaa ggtcattcaa aaggtcatcc
     3421 accggatcaa ttcccctgct cgcgcaggct gggtgccaag ctctcgggta acatcaaggc
     3481 ccgatccttg gagcccttgc cctcccgcac gatgatcgtg ccgtgatcga aatccagatc
     3541 cttgacccgc agttgcaaac cctcactgat ccgattcatt aatgcagctg gcacgacagg
     3601 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     3661 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     3721 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cggtacctgt
     3781 cgcggcaacg cgctaacaga cgtagtaaga accaccagca ttgtaatgct ggctaaagtc
     3841 actttcctga gctgtataac gatgagcgat tttacttttt ctggctatga attggcctgc
     3901 tttgtaacac actccggtct atcccgtagc gccgggcata tcctgtcgca atgtgcaaat
     3961 ctcgcggcaa caaccagtga atacttcatt cacaagcctc accgcctgat cgcggcagaa
     4021 actggttata gccaatcaac cgtcgttcgt gcattccgtg aagctgtaaa caaaggtatc
     4081 ctgtctgtag agattgttat cggcgatcac cgtgaacgtc gcgctaacct gtaccggttt
     4141 acaccatcct ttttggcctt cgcacaacaa gccaaaaatg cgctgataga aagcaaatta
     4201 aagatctctt cagcggcaac caaggttaaa gctgttctcg ctaagacatt ggctttattt
     4261 aattttttat ccacaccccc atgtcaaaat gataccccct ccccctgtca ggatgacgtg
     4321 gcaataaaga ataagaagtc acaagttaaa aaaacaaaaa gatcagtttc cggcggtgcc
     4381 ggaacaacca gcctcaaaaa attgacttca tggatcgcta aggcaaaagc aaaggctgac
     4441 aatctgcggt tatccaaaaa acgcactcaa aaacatgagt tcaagcagaa agtagaggcg
     4501 gctgcgcgga aatatgctta cctgaagaac aagcgttcgc ctgatattgg cgggatatca
     4561 aacttcgata acctaccgca ttgcatgacg gtaaacgaag ctcttaatgc ggttttagcc
     4621 aaaaataaag ataacgaaca atggggtata ccggcaggat tcagagggta atgaattgct
     4681 ctaattataa ccatgcatac tttcaacacc tctagtttgc catgaggcaa actcataggt
     4741 gtcctggtaa gaggacactg ttgccaaaac tggacgcccc attattgcaa ttaataaaca
     4801 actaacggac aattctacct aacaataagt ggagttgcgg ccggccgcga tccgctagca
     4861 aagccacgtt gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc
     4921 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     4981 tcaacgggaa acgtcttgct cgcgcccgcg attaaattcc aacatggatg ctgatttata
     5041 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta
     5101 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     5161 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     5221 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa
     5281 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     5341 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     5401 tcgcgtattt cgcctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     5461 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     5521 actgttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     5581 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     5641 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     5701 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     5761 tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcgagc
     5821 tctgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     5881 tatcacgagg cagaatttca gataaaaaaa atccttagct ttcgctaagg atgatttctg
     5941 cacctgcacc agtcagtaaa acgacggcca gtagtcaaaa gcctccgacc ggaggctttt
     6001 gacttggttc aggtggagtg ggag
//
