LOCUS       Exported                6570 bp DNA     circular SYN 02-JUL-2021
DEFINITION  BBF10K_000527 in pOpen_v4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6570)
  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..6570
                     /organism="BBF10K_000527 in pOpen_v4"
     primer_bind     21..51
                     /label=Gibson-for
     regulatory      46..55
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             52..2076
                     /codon_start=1
                     /product="anti-CD19 chimeric antigen receptor, 
                     intracellular CD28z"
                     /label=CD19CAR-CD28z
                     /translation="METDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRA
                     SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA
                     TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVT
                     CTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFL
                     KMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPPCPAPEFLGG
                     PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF
                     NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ
                     EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK
                     SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIF
                     WVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGRVKFSRSADAPAY
                     QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
                     EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR"
     sig_peptide     52..114
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
     primer_bind     complement(2077..2101)
                     /label=Gibson-rev
     terminator      2220..2267
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2272..2288)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2447..2697
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(2700..3455)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      3546..3765
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     protein_bind    4185..4206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4221..4251
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4259..4275
                     /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     4283..4299
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4372..5217
                     /codon_start=1
                     /label=repL
                     /translation="MLAKVTFLSCITMSDFTFSGYELACFVTHSGLSRSAGHILSQCAN
                     LAATTSEYFIHKPHRLIAAETGYSQSTVVRAFREAVNKGILSVEIVIGDHRERRANLYR
                     FTPSFLAFAQQAKNALIESKLKISSAATKVKAVLAKTLALFNFLSTPPCQNDTPSPCQD
                     DVAIKNKKSQVKKTKRSVSGGAGTTSLKKLTSWIAKAKAKADNLRLSKKRTQKHEFKQK
                     VEAAARKYAYLKNKRSPDIGGISNFDNLPHCMTVNEALNAVLAKNKDNEQWGIPAGFRG
                     "
     misc_feature    5325..5476
                     /label=nnpp promoters
     CDS             5516..6331
                     /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(6441..6484)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     6502..6518
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      6519..6550
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 aagaattact gacccctcgg cagactgagt cgcccggggg gatccgccac catggagaca
       61 gacacactcc tgctatgggt gctgctgctc tgggttccag gttccacagg tgacatccag
      121 atgacacaga ctacatcctc cctgtctgcc tctctgggag acagagtcac catcagttgc
      181 agggcaagtc aggacattag taaatattta aattggtatc agcagaaacc agatggaact
      241 gttaaactcc tgatctacca tacatcaaga ttacactcag gagtcccatc aaggttcagt
      301 ggcagtgggt ctggaacaga ttattctctc accattagca acctggagca agaagatatt
      361 gccacttact tttgccaaca gggtaatacg cttccgtaca cgttcggagg ggggaccaag
      421 ctggagatca caggtggcgg tggctcgggc ggtggtgggt cgggtggcgg cggatctgag
      481 gtgaaactgc aggagtcagg acctggcctg gtggcgccct cacagagcct gtccgtcaca
      541 tgcactgtct caggggtctc attacccgac tatggtgtaa gctggattcg ccagcctcca
      601 cgaaagggtc tggagtggct gggagtaata tggggtagtg aaaccacata ctataattca
      661 gctctcaaat ccagactgac catcatcaag gacaactcca agagccaagt tttcttaaaa
      721 atgaacagtc tgcaaactga tgacacagcc atttactact gtgccaaaca ttattactac
      781 ggtggtagct atgctatgga ctactggggc caaggaacct cagtcaccgt ctcctcagag
      841 agcaagtacg gccctccctg ccccccttgc cctgcccccg agttcctggg cggacccagc
      901 gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagccggac ccccgaggtg
      961 acctgcgtgg tggtggacgt gagccaggaa gatcccgagg tccagttcaa ttggtacgtg
     1021 gacggcgtgg aagtgcacaa cgccaagacc aagcccagag aggaacagtt caacagcacc
     1081 taccgggtgg tgtctgtgct gaccgtgctg caccaggact ggctgaacgg caaagaatac
     1141 aagtgcaagg tgtccaacaa gggcctgccc agcagcatcg aaaagaccat cagcaaggcc
     1201 aagggccagc ctcgcgagcc ccaggtgtac accctgcctc cctcccagga agagatgacc
     1261 aagaaccagg tgtccctgac ctgcctggtg aagggcttct accccagcga catcgccgtg
     1321 gagtgggaga gcaacggcca gcctgagaac aactacaaga ccacccctcc cgtgctggac
     1381 agcgacggca gcttcttcct gtacagccgg ctgaccgtgg acaagagccg gtggcaggaa
     1441 ggcaacgtct ttagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag
     1501 agcctgagcc tgtccctggg caagatgttc tgggtgctgg tggtggtggg cggggtgctg
     1561 gcctgctaca gcctgctggt gacagtggcc ttcatcatct tttgggtgcg gagcaagcgg
     1621 agcagaggcg gccacagcga ctacatgaac atgaccccca gacggcctgg ccccacccgg
     1681 aagcactacc agccctacgc cccacccagg gactttgccg cctaccggtc cggcggaggg
     1741 cgggtgaagt tcagcagaag cgccgacgcc cctgcctacc agcagggcca gaatcagctg
     1801 tacaacgagc tgaacctggg cagaagggaa gagtacgacg tcctggataa gcggagaggc
     1861 cgggaccctg agatgggcgg caagcctcgg cggaagaacc cccaggaagg cctgtataac
     1921 gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg
     1981 aggcggggca agggccacga cggcctgtat cagggcctgt ccaccgccac caaggatacc
     2041 tacgacgccc tgcacatgca ggccctgccc ccaaggggat ccgccaccaa cttcagcctg
     2101 ctaggtgatc tgacgaatgt ccttaaaaat ccccgctggc acttagggat aacagggtaa
     2161 tgctactcag gggtttcgca gtcgctgagg tgtcaatcgt cggagccgct gagcaataac
     2221 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgcat ggtcatagct
     2281 gtttcctgag agcttggcag gtgtcgcgta tgcatcgatc acgtgtccac gttctttaat
     2341 agtggactct tgttccaaac tggaacaaca ctcggatcga tccggcgcgc accgtgggaa
     2401 aaactccagg tagaggtaca cacgcggata gccaattcag agtaataaac tgtgataatc
     2461 aaccctcatc aatgatgacg aactaacccc cgatatcagg tcacatgacg aagggaaaga
     2521 gaaggaaatc aactgtgaca aactgccctc aaatttggct tccttaaaaa ttacagttca
     2581 aaaagtatga gaaaatccat gcaggctgaa ggaaacagca aaactgtgac aaattaccct
     2641 cagtaggtca gaacaaatgt gacgaaccac cctcaaatct gtgacagata accctcagac
     2701 tatcctgtcg tcatggaagt gatatcgcgg aaggaaaata cgatatgagt cgtctggcgg
     2761 cctttctttt tctcaatgta tgagaggcgc attggagttc tgctgttgat ctcattaaca
     2821 cagacttgca ggaagcggcg gcggaagtca ggcatacgct ggtaactttg aggcagctgg
     2881 taacgctcta tgatccagtc gattttcaga gacacgatgc ctgagccatc cggcttacga
     2941 tactgacaca gggattcgta taaacgcatg gcatacggat tggtgatttc ttttgtttca
     3001 ctaagccgaa actgcgtaaa ccggttctgt aacccgataa agaagggaat gagatatggg
     3061 ttgatatgta cactgtaaag ccctctggat ggactgtgcg cacgtttgat aaaccaagga
     3121 aaagattcat agcctttttc atcaccggca tcctcttcag ggcgataaaa aaccacttcc
     3181 ttccccgcga aactcttcaa tgcctgccgt atatccttac tggcttccgc agaggtcaat
     3241 ccgaatattt cagcatattt agcaacatgg atctcgcaga taccgtcatg ttcctgtagg
     3301 gtgccatcag attttctgat ctggtcaacg aacagataca gcatacgttt ttgatcccgg
     3361 gagagactat atgccgcctc agtgaggtcg tttgactgga cgattcgcgg gctattttta
     3421 cgtttcttgt gattgataac cgctgtttcc gccatgacag atccatgtga agtgtgacaa
     3481 gtttttagat tgtcacacta aataaaaaag agtcaataag cagggataac tttgtgaaaa
     3541 aacagcttct tctgagggca atttgtcaca gggttaaggg caatttgtca cagacaggac
     3601 tgtcatttga gggtgatttg tcacactgaa agggcaattt gtcacaacac cttctgtaga
     3661 accagcatgg ataaaggcct acaaggcgct ctaaaaaaga agatctaaaa actataaaaa
     3721 aaataattat aaaaatatcc ccgtggataa gtggataacc ccaagggaag ttttttcagg
     3781 catcgtgtgt aagcagaata tataagtgct gttccctggt gcttcctcgc tcactcgaaa
     3841 ttcccgggga tagctttatg cttgtaaacc gttttgtgaa aaaattttta aaataaaaaa
     3901 ggggacctct agggtcccca attaattagt aatataatct attaaaggtc attcaaaagg
     3961 tcatccaccg gatcaattcc cctgctcgcg caggctgggt gccaagctct cgggtaacat
     4021 caaggcccga tccttggagc ccttgccctc ccgcacgatg atcgtgccgt gatcgaaatc
     4081 cagatccttg acccgcagtt gcaaaccctc actgatccga ttcattaatg cagctggcac
     4141 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     4201 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     4261 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcggt
     4321 acctgtcgcg gcaacgcgct aacagacgta gtaagaacca ccagcattgt aatgctggct
     4381 aaagtcactt tcctgagctg tataacgatg agcgatttta ctttttctgg ctatgaattg
     4441 gcctgctttg taacacactc cggtctatcc cgtagcgccg ggcatatcct gtcgcaatgt
     4501 gcaaatctcg cggcaacaac cagtgaatac ttcattcaca agcctcaccg cctgatcgcg
     4561 gcagaaactg gttatagcca atcaaccgtc gttcgtgcat tccgtgaagc tgtaaacaaa
     4621 ggtatcctgt ctgtagagat tgttatcggc gatcaccgtg aacgtcgcgc taacctgtac
     4681 cggtttacac catccttttt ggccttcgca caacaagcca aaaatgcgct gatagaaagc
     4741 aaattaaaga tctcttcagc ggcaaccaag gttaaagctg ttctcgctaa gacattggct
     4801 ttatttaatt ttttatccac acccccatgt caaaatgata ccccctcccc ctgtcaggat
     4861 gacgtggcaa taaagaataa gaagtcacaa gttaaaaaaa caaaaagatc agtttccggc
     4921 ggtgccggaa caaccagcct caaaaaattg acttcatgga tcgctaaggc aaaagcaaag
     4981 gctgacaatc tgcggttatc caaaaaacgc actcaaaaac atgagttcaa gcagaaagta
     5041 gaggcggctg cgcggaaata tgcttacctg aagaacaagc gttcgcctga tattggcggg
     5101 atatcaaact tcgataacct accgcattgc atgacggtaa acgaagctct taatgcggtt
     5161 ttagccaaaa ataaagataa cgaacaatgg ggtataccgg caggattcag agggtaatga
     5221 attgctctaa ttataaccat gcatactttc aacacctcta gtttgccatg aggcaaactc
     5281 ataggtgtcc tggtaagagg acactgttgc caaaactgga cgccccatta ttgcaattaa
     5341 taaacaacta acggacaatt ctacctaaca ataagtggag ttgcggccgg ccgcgatccg
     5401 ctagcaaagc cacgttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     5461 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     5521 ccatattcaa cgggaaacgt cttgctcgcg cccgcgatta aattccaaca tggatgctga
     5581 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     5641 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     5701 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     5761 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     5821 cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     5881 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     5941 cagcgatcgc gtatttcgcc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     6001 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     6061 gcataaactg ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     6121 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     6181 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     6241 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     6301 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg
     6361 gcgagctctg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     6421 taggcgtatc acgaggcaga atttcagata aaaaaaatcc ttagctttcg ctaaggatga
     6481 tttctgcacc tgcaccagtc agtaaaacga cggccagtag tcaaaagcct ccgaccggag
     6541 gcttttgact tggttcaggt ggagtgggag
//
