LOCUS       Exported                5892 bp DNA     circular SYN 02-JUL-2021
DEFINITION  BBF10K_000526 in pOpen_v4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5892)
  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..5892
                     /organism="BBF10K_000526 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..1398
                     /codon_start=1
                     /product="anti-streptavidin chimeric antigen receptor, 
                     intracellular 2B4"
                     /label=StrepCAR-2B4
                     /translation="METDTLLLWVLLLWVPGSTGGAEAGITGTWYNQHGSTFTVTAGAD
                     GNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGA
                     EARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSTTTPAPRPPTPAPTIASQPLS
                     LRPEACRPAAGGAVHTRGLDFACDMFWVLVVVGGVLACYSLLVTVAFIIFWVWRRKRKE
                     KQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTL
                     YSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSCGRVK
                     FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL
                     QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR"
     CDS             complement(949..960)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1399..1423)
                     /label=Gibson-rev
     terminator      1542..1589
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1594..1610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1769..2019
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(2022..2777)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      2868..3087
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     protein_bind    3507..3528
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3543..3573
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3581..3597
                     /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     3605..3621
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3694..4539
                     /codon_start=1
                     /label=repL
                     /translation="MLAKVTFLSCITMSDFTFSGYELACFVTHSGLSRSAGHILSQCAN
                     LAATTSEYFIHKPHRLIAAETGYSQSTVVRAFREAVNKGILSVEIVIGDHRERRANLYR
                     FTPSFLAFAQQAKNALIESKLKISSAATKVKAVLAKTLALFNFLSTPPCQNDTPSPCQD
                     DVAIKNKKSQVKKTKRSVSGGAGTTSLKKLTSWIAKAKAKADNLRLSKKRTQKHEFKQK
                     VEAAARKYAYLKNKRSPDIGGISNFDNLPHCMTVNEALNAVLAKNKDNEQWGIPAGFRG
                     "
     misc_feature    4647..4798
                     /label=nnpp promoters
     CDS             4838..5653
                     /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(5763..5806)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     5824..5840
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      5841..5872
                     /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 tgggtgtgga ggagaaagag gaaggagaag
      721 cagtcagaga ccagtcccaa ggaatttttg acaatttacg aagatgtcaa ggatctgaaa
      781 accaggagaa atcacgagca ggagcagact tttcctggag gggggagcac catctactct
      841 atgatccagt cccagtcttc tgctcccacg tcacaagaac ctgcatatac attatattca
      901 ttaattcagc cttccaggaa gtctgggtcc aggaagagga accacagccc ttccttcaat
      961 agcactatct atgaagtgat tggaaagagt caacctaaag cccagaaccc tgctcgattg
     1021 agccgcaaag agctggagaa ctttgatgtt tattcctgtg ggcgggtgaa gttcagcaga
     1081 agcgccgacg cccctgccta ccagcagggc cagaatcagc tgtacaacga gctgaacctg
     1141 ggcagaaggg aagagtacga cgtcctggat aagcggagag gccgggaccc tgagatgggc
     1201 ggcaagcctc ggcggaagaa cccccaggaa ggcctgtata acgaactgca gaaagacaag
     1261 atggccgagg cctacagcga gatcggcatg aagggcgagc ggaggcgggg caagggccac
     1321 gacggcctgt atcagggcct gtccaccgcc accaaggata cctacgacgc cctgcacatg
     1381 caggccctgc ccccaagggg atccgccacc aacttcagcc tgctagggct aattaccatc
     1441 agcgcacagc cgcccctagt ataagtaggg ataacagggt aatgccgaga gctgaggata
     1501 acgccgctga ggtgtcaatc gtcggagccg ctgagcaata actagcataa ccccttgggg
     1561 cctctaaacg ggtcttgagg ggttttttgc atggtcatag ctgtttcctg agagcttggc
     1621 aggtgtcgcg tatgcatcga tcacgtgtcc acgttcttta atagtggact cttgttccaa
     1681 actggaacaa cactcggatc gatccggcgc gcaccgtggg aaaaactcca ggtagaggta
     1741 cacacgcgga tagccaattc agagtaataa actgtgataa tcaaccctca tcaatgatga
     1801 cgaactaacc cccgatatca ggtcacatga cgaagggaaa gagaaggaaa tcaactgtga
     1861 caaactgccc tcaaatttgg cttccttaaa aattacagtt caaaaagtat gagaaaatcc
     1921 atgcaggctg aaggaaacag caaaactgtg acaaattacc ctcagtaggt cagaacaaat
     1981 gtgacgaacc accctcaaat ctgtgacaga taaccctcag actatcctgt cgtcatggaa
     2041 gtgatatcgc ggaaggaaaa tacgatatga gtcgtctggc ggcctttctt tttctcaatg
     2101 tatgagaggc gcattggagt tctgctgttg atctcattaa cacagacttg caggaagcgg
     2161 cggcggaagt caggcatacg ctggtaactt tgaggcagct ggtaacgctc tatgatccag
     2221 tcgattttca gagacacgat gcctgagcca tccggcttac gatactgaca cagggattcg
     2281 tataaacgca tggcatacgg attggtgatt tcttttgttt cactaagccg aaactgcgta
     2341 aaccggttct gtaacccgat aaagaaggga atgagatatg ggttgatatg tacactgtaa
     2401 agccctctgg atggactgtg cgcacgtttg ataaaccaag gaaaagattc atagcctttt
     2461 tcatcaccgg catcctcttc agggcgataa aaaaccactt ccttccccgc gaaactcttc
     2521 aatgcctgcc gtatatcctt actggcttcc gcagaggtca atccgaatat ttcagcatat
     2581 ttagcaacat ggatctcgca gataccgtca tgttcctgta gggtgccatc agattttctg
     2641 atctggtcaa cgaacagata cagcatacgt ttttgatccc gggagagact atatgccgcc
     2701 tcagtgaggt cgtttgactg gacgattcgc gggctatttt tacgtttctt gtgattgata
     2761 accgctgttt ccgccatgac agatccatgt gaagtgtgac aagtttttag attgtcacac
     2821 taaataaaaa agagtcaata agcagggata actttgtgaa aaaacagctt cttctgaggg
     2881 caatttgtca cagggttaag ggcaatttgt cacagacagg actgtcattt gagggtgatt
     2941 tgtcacactg aaagggcaat ttgtcacaac accttctgta gaaccagcat ggataaaggc
     3001 ctacaaggcg ctctaaaaaa gaagatctaa aaactataaa aaaaataatt ataaaaatat
     3061 ccccgtggat aagtggataa ccccaaggga agttttttca ggcatcgtgt gtaagcagaa
     3121 tatataagtg ctgttccctg gtgcttcctc gctcactcga aattcccggg gatagcttta
     3181 tgcttgtaaa ccgttttgtg aaaaaatttt taaaataaaa aaggggacct ctagggtccc
     3241 caattaatta gtaatataat ctattaaagg tcattcaaaa ggtcatccac cggatcaatt
     3301 cccctgctcg cgcaggctgg gtgccaagct ctcgggtaac atcaaggccc gatccttgga
     3361 gcccttgccc tcccgcacga tgatcgtgcc gtgatcgaaa tccagatcct tgacccgcag
     3421 ttgcaaaccc tcactgatcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     3481 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     3541 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     3601 cacacaggaa acagctatga ccatgattac gccaagcgcg gtacctgtcg cggcaacgcg
     3661 ctaacagacg tagtaagaac caccagcatt gtaatgctgg ctaaagtcac tttcctgagc
     3721 tgtataacga tgagcgattt tactttttct ggctatgaat tggcctgctt tgtaacacac
     3781 tccggtctat cccgtagcgc cgggcatatc ctgtcgcaat gtgcaaatct cgcggcaaca
     3841 accagtgaat acttcattca caagcctcac cgcctgatcg cggcagaaac tggttatagc
     3901 caatcaaccg tcgttcgtgc attccgtgaa gctgtaaaca aaggtatcct gtctgtagag
     3961 attgttatcg gcgatcaccg tgaacgtcgc gctaacctgt accggtttac accatccttt
     4021 ttggccttcg cacaacaagc caaaaatgcg ctgatagaaa gcaaattaaa gatctcttca
     4081 gcggcaacca aggttaaagc tgttctcgct aagacattgg ctttatttaa ttttttatcc
     4141 acacccccat gtcaaaatga taccccctcc ccctgtcagg atgacgtggc aataaagaat
     4201 aagaagtcac aagttaaaaa aacaaaaaga tcagtttccg gcggtgccgg aacaaccagc
     4261 ctcaaaaaat tgacttcatg gatcgctaag gcaaaagcaa aggctgacaa tctgcggtta
     4321 tccaaaaaac gcactcaaaa acatgagttc aagcagaaag tagaggcggc tgcgcggaaa
     4381 tatgcttacc tgaagaacaa gcgttcgcct gatattggcg ggatatcaaa cttcgataac
     4441 ctaccgcatt gcatgacggt aaacgaagct cttaatgcgg ttttagccaa aaataaagat
     4501 aacgaacaat ggggtatacc ggcaggattc agagggtaat gaattgctct aattataacc
     4561 atgcatactt tcaacacctc tagtttgcca tgaggcaaac tcataggtgt cctggtaaga
     4621 ggacactgtt gccaaaactg gacgccccat tattgcaatt aataaacaac taacggacaa
     4681 ttctacctaa caataagtgg agttgcggcc ggccgcgatc cgctagcaaa gccacgttgt
     4741 gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa
     4801 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     4861 gtcttgctcg cgcccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     4921 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     4981 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     5041 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     5101 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca
     5161 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     5221 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     5281 cctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     5341 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac tgttgccatt
     5401 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     5461 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     5521 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     5581 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     5641 tgagtttttc taatcagaat tggttaattg gttgtaacac tggcgagctc tgccacctga
     5701 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggca
     5761 gaatttcaga taaaaaaaat ccttagcttt cgctaaggat gatttctgca cctgcaccag
     5821 tcagtaaaac gacggccagt agtcaaaagc ctccgaccgg aggcttttga cttggttcag
     5881 gtggagtggg ag
//
