LOCUS       Exported                5658 bp DNA     circular SYN 02-JUL-2021
DEFINITION  BBF10K_000524 in pOpen_v4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5658)
  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..5658
                     /organism="BBF10K_000524 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..1164
                     /codon_start=1
                     /product="anti-streptavidin chimeric antigen receptor, 
                     intracellular CD28z"
                     /label=StrepCAR-CD28z
                     /translation="METDTLLLWVLLLWVPGSTGGAEAGITGTWYNQHGSTFTVTAGAD
                     GNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGA
                     EARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSTTTPAPRPPTPAPTIASQPLS
                     LRPEACRPAAGGAVHTRGLDFACDMFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRG
                     GHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGRVKFSRSADAPAYQQGQNQLYN
                     ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR
                     RGKGHDGLYQGLSTATKDTYDALHMQALPPR"
     primer_bind     complement(1165..1189)
                     /label=Gibson-rev
     terminator      1308..1355
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1360..1376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1535..1785
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(1788..2543)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      2634..2853
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     protein_bind    3273..3294
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3309..3339
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3347..3363
                     /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     3371..3387
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3460..4305
                     /codon_start=1
                     /label=repL
                     /translation="MLAKVTFLSCITMSDFTFSGYELACFVTHSGLSRSAGHILSQCAN
                     LAATTSEYFIHKPHRLIAAETGYSQSTVVRAFREAVNKGILSVEIVIGDHRERRANLYR
                     FTPSFLAFAQQAKNALIESKLKISSAATKVKAVLAKTLALFNFLSTPPCQNDTPSPCQD
                     DVAIKNKKSQVKKTKRSVSGGAGTTSLKKLTSWIAKAKAKADNLRLSKKRTQKHEFKQK
                     VEAAARKYAYLKNKRSPDIGGISNFDNLPHCMTVNEALNAVLAKNKDNEQWGIPAGFRG
                     "
     misc_feature    4413..4564
                     /label=nnpp promoters
     CDS             4604..5419
                     /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(5529..5572)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     5590..5606
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      5607..5638
                     /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 tggcgcagag
      121 gcgggtatca ccgggacatg gtacaaccaa cacggaagca catttacagt caccgctgga
      181 gcagacggga atctgaccgg acagtacgag aacagggctc aggggacagg ttgtcagaac
      241 agtccgtata ctctgactgg gaggtacaat ggcacgaagc tggagtggcg agtcgagtgg
      301 aataattcca cggaaaactg tcacagtaga acagagtgga ggggacagta ccagggggga
      361 gcagaggccc ggatcaacac ccaatggaac ttgacatatg aaggcgggtc aggccccgcg
      421 acagagcaag gacaggatac attcacgaag gtcaagccaa gcgcagcctc tggctctacc
      481 acaactccag ctccccggcc ccctactcct gctccaacca ttgcctcaca gccactgagc
      541 ctgcggcccg aagcttgtag acctgctgct ggaggagctg tgcataccag aggcctggac
      601 ttcgcctgcg atatgttctg ggtgctggtg gtggtgggcg gggtgctggc ctgctacagc
      661 ctgctggtga cagtggcctt catcatcttt tgggtgcgga gcaagcggag cagaggcggc
      721 cacagcgact acatgaacat gacccccaga cggcctggcc ccacccggaa gcactaccag
      781 ccctacgccc cacccaggga ctttgccgcc taccggtccg gcggagggcg ggtgaagttc
      841 agcagaagcg ccgacgcccc tgcctaccag cagggccaga atcagctgta caacgagctg
      901 aacctgggca gaagggaaga gtacgacgtc ctggataagc ggagaggccg ggaccctgag
      961 atgggcggca agcctcggcg gaagaacccc caggaaggcc tgtataacga actgcagaaa
     1021 gacaagatgg ccgaggccta cagcgagatc ggcatgaagg gcgagcggag gcggggcaag
     1081 ggccacgacg gcctgtatca gggcctgtcc accgccacca aggataccta cgacgccctg
     1141 cacatgcagg ccctgccccc aaggggatcc gccaccaact tcagcctgct agctcaatct
     1201 aacagtgggg ttatctcttt acgagcactc gtagggataa cagggtaata gtacatgcca
     1261 ctccaagggt cgctgaggtg tcaatcgtcg gagccgctga gcaataacta gcataacccc
     1321 ttggggcctc taaacgggtc ttgaggggtt ttttgcatgg tcatagctgt ttcctgagag
     1381 cttggcaggt gtcgcgtatg catcgatcac gtgtccacgt tctttaatag tggactcttg
     1441 ttccaaactg gaacaacact cggatcgatc cggcgcgcac cgtgggaaaa actccaggta
     1501 gaggtacaca cgcggatagc caattcagag taataaactg tgataatcaa ccctcatcaa
     1561 tgatgacgaa ctaacccccg atatcaggtc acatgacgaa gggaaagaga aggaaatcaa
     1621 ctgtgacaaa ctgccctcaa atttggcttc cttaaaaatt acagttcaaa aagtatgaga
     1681 aaatccatgc aggctgaagg aaacagcaaa actgtgacaa attaccctca gtaggtcaga
     1741 acaaatgtga cgaaccaccc tcaaatctgt gacagataac cctcagacta tcctgtcgtc
     1801 atggaagtga tatcgcggaa ggaaaatacg atatgagtcg tctggcggcc tttctttttc
     1861 tcaatgtatg agaggcgcat tggagttctg ctgttgatct cattaacaca gacttgcagg
     1921 aagcggcggc ggaagtcagg catacgctgg taactttgag gcagctggta acgctctatg
     1981 atccagtcga ttttcagaga cacgatgcct gagccatccg gcttacgata ctgacacagg
     2041 gattcgtata aacgcatggc atacggattg gtgatttctt ttgtttcact aagccgaaac
     2101 tgcgtaaacc ggttctgtaa cccgataaag aagggaatga gatatgggtt gatatgtaca
     2161 ctgtaaagcc ctctggatgg actgtgcgca cgtttgataa accaaggaaa agattcatag
     2221 cctttttcat caccggcatc ctcttcaggg cgataaaaaa ccacttcctt ccccgcgaaa
     2281 ctcttcaatg cctgccgtat atccttactg gcttccgcag aggtcaatcc gaatatttca
     2341 gcatatttag caacatggat ctcgcagata ccgtcatgtt cctgtagggt gccatcagat
     2401 tttctgatct ggtcaacgaa cagatacagc atacgttttt gatcccggga gagactatat
     2461 gccgcctcag tgaggtcgtt tgactggacg attcgcgggc tatttttacg tttcttgtga
     2521 ttgataaccg ctgtttccgc catgacagat ccatgtgaag tgtgacaagt ttttagattg
     2581 tcacactaaa taaaaaagag tcaataagca gggataactt tgtgaaaaaa cagcttcttc
     2641 tgagggcaat ttgtcacagg gttaagggca atttgtcaca gacaggactg tcatttgagg
     2701 gtgatttgtc acactgaaag ggcaatttgt cacaacacct tctgtagaac cagcatggat
     2761 aaaggcctac aaggcgctct aaaaaagaag atctaaaaac tataaaaaaa ataattataa
     2821 aaatatcccc gtggataagt ggataacccc aagggaagtt ttttcaggca tcgtgtgtaa
     2881 gcagaatata taagtgctgt tccctggtgc ttcctcgctc actcgaaatt cccggggata
     2941 gctttatgct tgtaaaccgt tttgtgaaaa aatttttaaa ataaaaaagg ggacctctag
     3001 ggtccccaat taattagtaa tataatctat taaaggtcat tcaaaaggtc atccaccgga
     3061 tcaattcccc tgctcgcgca ggctgggtgc caagctctcg ggtaacatca aggcccgatc
     3121 cttggagccc ttgccctccc gcacgatgat cgtgccgtga tcgaaatcca gatccttgac
     3181 ccgcagttgc aaaccctcac tgatccgatt cattaatgca gctggcacga caggtttccc
     3241 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     3301 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     3361 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcggtac ctgtcgcggc
     3421 aacgcgctaa cagacgtagt aagaaccacc agcattgtaa tgctggctaa agtcactttc
     3481 ctgagctgta taacgatgag cgattttact ttttctggct atgaattggc ctgctttgta
     3541 acacactccg gtctatcccg tagcgccggg catatcctgt cgcaatgtgc aaatctcgcg
     3601 gcaacaacca gtgaatactt cattcacaag cctcaccgcc tgatcgcggc agaaactggt
     3661 tatagccaat caaccgtcgt tcgtgcattc cgtgaagctg taaacaaagg tatcctgtct
     3721 gtagagattg ttatcggcga tcaccgtgaa cgtcgcgcta acctgtaccg gtttacacca
     3781 tcctttttgg ccttcgcaca acaagccaaa aatgcgctga tagaaagcaa attaaagatc
     3841 tcttcagcgg caaccaaggt taaagctgtt ctcgctaaga cattggcttt atttaatttt
     3901 ttatccacac ccccatgtca aaatgatacc ccctccccct gtcaggatga cgtggcaata
     3961 aagaataaga agtcacaagt taaaaaaaca aaaagatcag tttccggcgg tgccggaaca
     4021 accagcctca aaaaattgac ttcatggatc gctaaggcaa aagcaaaggc tgacaatctg
     4081 cggttatcca aaaaacgcac tcaaaaacat gagttcaagc agaaagtaga ggcggctgcg
     4141 cggaaatatg cttacctgaa gaacaagcgt tcgcctgata ttggcgggat atcaaacttc
     4201 gataacctac cgcattgcat gacggtaaac gaagctctta atgcggtttt agccaaaaat
     4261 aaagataacg aacaatgggg tataccggca ggattcagag ggtaatgaat tgctctaatt
     4321 ataaccatgc atactttcaa cacctctagt ttgccatgag gcaaactcat aggtgtcctg
     4381 gtaagaggac actgttgcca aaactggacg ccccattatt gcaattaata aacaactaac
     4441 ggacaattct acctaacaat aagtggagtt gcggccggcc gcgatccgct agcaaagcca
     4501 cgttgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac
     4561 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
     4621 ggaaacgtct tgctcgcgcc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     4681 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa
     4741 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     4801 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     4861 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc
     4921 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     4981 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     5041 atttcgcctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     5101 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactgtt
     5161 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     5221 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     5281 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     5341 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     5401 gctcgatgag tttttctaat cagaattggt taattggttg taacactggc gagctctgcc
     5461 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     5521 gaggcagaat ttcagataaa aaaaatcctt agctttcgct aaggatgatt tctgcacctg
     5581 caccagtcag taaaacgacg gccagtagtc aaaagcctcc gaccggaggc ttttgacttg
     5641 gttcaggtgg agtgggag
//
