LOCUS       Exported                5652 bp DNA     circular SYN 02-JUL-2021
DEFINITION  BBF10K_000525 in pOpen_v4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5652)
  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..5652
                     /organism="BBF10K_000525 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..1158
                     /codon_start=1
                     /product="anti-streptavidin chimeric antigen receptor, 
                     intracellular 4-1BBz"
                     /label=StrepCAR-41BBz
                     /translation="METDTLLLWVLLLWVPGSTGGAEAGITGTWYNQHGSTFTVTAGAD
                     GNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGA
                     EARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSTTTPAPRPPTPAPTIASQPLS
                     LRPEACRPAAGGAVHTRGLDFACDMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKL
                     LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL
                     NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG
                     KGHDGLYQGLSTATKDTYDALHMQALPPR"
     primer_bind     complement(1159..1183)
                     /label=Gibson-rev
     terminator      1302..1349
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1354..1370)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1529..1779
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(1782..2537)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      2628..2847
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     protein_bind    3267..3288
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3303..3333
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3341..3357
                     /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     3365..3381
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3454..4299
                     /codon_start=1
                     /label=repL
                     /translation="MLAKVTFLSCITMSDFTFSGYELACFVTHSGLSRSAGHILSQCAN
                     LAATTSEYFIHKPHRLIAAETGYSQSTVVRAFREAVNKGILSVEIVIGDHRERRANLYR
                     FTPSFLAFAQQAKNALIESKLKISSAATKVKAVLAKTLALFNFLSTPPCQNDTPSPCQD
                     DVAIKNKKSQVKKTKRSVSGGAGTTSLKKLTSWIAKAKAKADNLRLSKKRTQKHEFKQK
                     VEAAARKYAYLKNKRSPDIGGISNFDNLPHCMTVNEALNAVLAKNKDNEQWGIPAGFRG
                     "
     misc_feature    4407..4558
                     /label=nnpp promoters
     CDS             4598..5413
                     /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(5523..5566)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     5584..5600
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      5601..5632
                     /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 tgggtgaaac ggggcagaaa gaaactcctg
      721 tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt
      781 agctgccgat ttccagaaga agaagaagga ggatgtgaac tgcgggtgaa gttcagcaga
      841 agcgccgacg cccctgccta ccagcagggc cagaatcagc tgtacaacga gctgaacctg
      901 ggcagaaggg aagagtacga cgtcctggat aagcggagag gccgggaccc tgagatgggc
      961 ggcaagcctc ggcggaagaa cccccaggaa ggcctgtata acgaactgca gaaagacaag
     1021 atggccgagg cctacagcga gatcggcatg aagggcgagc ggaggcgggg caagggccac
     1081 gacggcctgt atcagggcct gtccaccgcc accaaggata cctacgacgc cctgcacatg
     1141 caggccctgc ccccaagggg atccgccacc aacttcagcc tgctaggaca ccagaccaat
     1201 gaagctgaat caaaggcgcc gttcttaggg ataacagggt aatgacaagg tctaggtgga
     1261 agtccgctga ggtgtcaatc gtcggagccg ctgagcaata actagcataa ccccttgggg
     1321 cctctaaacg ggtcttgagg ggttttttgc atggtcatag ctgtttcctg agagcttggc
     1381 aggtgtcgcg tatgcatcga tcacgtgtcc acgttcttta atagtggact cttgttccaa
     1441 actggaacaa cactcggatc gatccggcgc gcaccgtggg aaaaactcca ggtagaggta
     1501 cacacgcgga tagccaattc agagtaataa actgtgataa tcaaccctca tcaatgatga
     1561 cgaactaacc cccgatatca ggtcacatga cgaagggaaa gagaaggaaa tcaactgtga
     1621 caaactgccc tcaaatttgg cttccttaaa aattacagtt caaaaagtat gagaaaatcc
     1681 atgcaggctg aaggaaacag caaaactgtg acaaattacc ctcagtaggt cagaacaaat
     1741 gtgacgaacc accctcaaat ctgtgacaga taaccctcag actatcctgt cgtcatggaa
     1801 gtgatatcgc ggaaggaaaa tacgatatga gtcgtctggc ggcctttctt tttctcaatg
     1861 tatgagaggc gcattggagt tctgctgttg atctcattaa cacagacttg caggaagcgg
     1921 cggcggaagt caggcatacg ctggtaactt tgaggcagct ggtaacgctc tatgatccag
     1981 tcgattttca gagacacgat gcctgagcca tccggcttac gatactgaca cagggattcg
     2041 tataaacgca tggcatacgg attggtgatt tcttttgttt cactaagccg aaactgcgta
     2101 aaccggttct gtaacccgat aaagaaggga atgagatatg ggttgatatg tacactgtaa
     2161 agccctctgg atggactgtg cgcacgtttg ataaaccaag gaaaagattc atagcctttt
     2221 tcatcaccgg catcctcttc agggcgataa aaaaccactt ccttccccgc gaaactcttc
     2281 aatgcctgcc gtatatcctt actggcttcc gcagaggtca atccgaatat ttcagcatat
     2341 ttagcaacat ggatctcgca gataccgtca tgttcctgta gggtgccatc agattttctg
     2401 atctggtcaa cgaacagata cagcatacgt ttttgatccc gggagagact atatgccgcc
     2461 tcagtgaggt cgtttgactg gacgattcgc gggctatttt tacgtttctt gtgattgata
     2521 accgctgttt ccgccatgac agatccatgt gaagtgtgac aagtttttag attgtcacac
     2581 taaataaaaa agagtcaata agcagggata actttgtgaa aaaacagctt cttctgaggg
     2641 caatttgtca cagggttaag ggcaatttgt cacagacagg actgtcattt gagggtgatt
     2701 tgtcacactg aaagggcaat ttgtcacaac accttctgta gaaccagcat ggataaaggc
     2761 ctacaaggcg ctctaaaaaa gaagatctaa aaactataaa aaaaataatt ataaaaatat
     2821 ccccgtggat aagtggataa ccccaaggga agttttttca ggcatcgtgt gtaagcagaa
     2881 tatataagtg ctgttccctg gtgcttcctc gctcactcga aattcccggg gatagcttta
     2941 tgcttgtaaa ccgttttgtg aaaaaatttt taaaataaaa aaggggacct ctagggtccc
     3001 caattaatta gtaatataat ctattaaagg tcattcaaaa ggtcatccac cggatcaatt
     3061 cccctgctcg cgcaggctgg gtgccaagct ctcgggtaac atcaaggccc gatccttgga
     3121 gcccttgccc tcccgcacga tgatcgtgcc gtgatcgaaa tccagatcct tgacccgcag
     3181 ttgcaaaccc tcactgatcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     3241 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     3301 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     3361 cacacaggaa acagctatga ccatgattac gccaagcgcg gtacctgtcg cggcaacgcg
     3421 ctaacagacg tagtaagaac caccagcatt gtaatgctgg ctaaagtcac tttcctgagc
     3481 tgtataacga tgagcgattt tactttttct ggctatgaat tggcctgctt tgtaacacac
     3541 tccggtctat cccgtagcgc cgggcatatc ctgtcgcaat gtgcaaatct cgcggcaaca
     3601 accagtgaat acttcattca caagcctcac cgcctgatcg cggcagaaac tggttatagc
     3661 caatcaaccg tcgttcgtgc attccgtgaa gctgtaaaca aaggtatcct gtctgtagag
     3721 attgttatcg gcgatcaccg tgaacgtcgc gctaacctgt accggtttac accatccttt
     3781 ttggccttcg cacaacaagc caaaaatgcg ctgatagaaa gcaaattaaa gatctcttca
     3841 gcggcaacca aggttaaagc tgttctcgct aagacattgg ctttatttaa ttttttatcc
     3901 acacccccat gtcaaaatga taccccctcc ccctgtcagg atgacgtggc aataaagaat
     3961 aagaagtcac aagttaaaaa aacaaaaaga tcagtttccg gcggtgccgg aacaaccagc
     4021 ctcaaaaaat tgacttcatg gatcgctaag gcaaaagcaa aggctgacaa tctgcggtta
     4081 tccaaaaaac gcactcaaaa acatgagttc aagcagaaag tagaggcggc tgcgcggaaa
     4141 tatgcttacc tgaagaacaa gcgttcgcct gatattggcg ggatatcaaa cttcgataac
     4201 ctaccgcatt gcatgacggt aaacgaagct cttaatgcgg ttttagccaa aaataaagat
     4261 aacgaacaat ggggtatacc ggcaggattc agagggtaat gaattgctct aattataacc
     4321 atgcatactt tcaacacctc tagtttgcca tgaggcaaac tcataggtgt cctggtaaga
     4381 ggacactgtt gccaaaactg gacgccccat tattgcaatt aataaacaac taacggacaa
     4441 ttctacctaa caataagtgg agttgcggcc ggccgcgatc cgctagcaaa gccacgttgt
     4501 gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa
     4561 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     4621 gtcttgctcg cgcccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     4681 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     4741 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     4801 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     4861 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca
     4921 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     4981 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     5041 cctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     5101 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac tgttgccatt
     5161 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     5221 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     5281 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     5341 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     5401 tgagtttttc taatcagaat tggttaattg gttgtaacac tggcgagctc tgccacctga
     5461 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggca
     5521 gaatttcaga taaaaaaaat ccttagcttt cgctaaggat gatttctgca cctgcaccag
     5581 tcagtaaaac gacggccagt agtcaaaagc ctccgaccgg aggcttttga cttggttcag
     5641 gtggagtggg ag
//
