LOCUS       Exported                5328 bp DNA     circular SYN 02-JUL-2021
DEFINITION  BBF10K_000517 in pOpen_v4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5328)
  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..5328
                     /organism="BBF10K_000517 in pOpen_v4"
     primer_bind     21..51
                     /label=Gibson-for
     CDS             52..834
                     /codon_start=1
                     /product="CD154 cluster of differentiation 154"
                     /label=CD40L
                     /translation="MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVY
                     LHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEE
                     TKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTV
                     KRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQS
                     IHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL"
     primer_bind     complement(835..859)
                     /label=Gibson-rev
     terminator      978..1025
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1030..1046)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1205..1455
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(1458..2213)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      2304..2523
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     protein_bind    2943..2964
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2979..3009
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3017..3033
                     /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     3041..3057
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3130..3975
                     /codon_start=1
                     /label=repL
                     /translation="MLAKVTFLSCITMSDFTFSGYELACFVTHSGLSRSAGHILSQCAN
                     LAATTSEYFIHKPHRLIAAETGYSQSTVVRAFREAVNKGILSVEIVIGDHRERRANLYR
                     FTPSFLAFAQQAKNALIESKLKISSAATKVKAVLAKTLALFNFLSTPPCQNDTPSPCQD
                     DVAIKNKKSQVKKTKRSVSGGAGTTSLKKLTSWIAKAKAKADNLRLSKKRTQKHEFKQK
                     VEAAARKYAYLKNKRSPDIGGISNFDNLPHCMTVNEALNAVLAKNKDNEQWGIPAGFRG
                     "
     misc_feature    4083..4234
                     /label=nnpp promoters
     CDS             4274..5089
                     /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(5199..5242)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     5260..5276
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      5277..5308
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 aagaattact gacccctcgg cagactgagt cgcccggggg gatccgccac catgatcgag
       61 acctacaacc agaccagccc cagaagcgcc gccaccggcc tgcccatcag catgaagatc
      121 ttcatgtacc tgctgaccgt gttcctgatc acccagatga tcggcagcgc cctgttcgcc
      181 gtgtacctgc acagaagact ggacaagatc gaggacgaga gaaacctgca cgaggacttc
      241 gtgttcatga agaccatcca gagatgcaac accggcgaga gaagcctgag cctgctgaac
      301 tgcgaggaga tcaagagcca gttcgagggc ttcgtgaagg acatcatgct gaacaaggag
      361 gagaccaaga aggagaacag cttcgagatg cagaagggcg accagaaccc ccagatcgcc
      421 gcccacgtga tcagcgaggc cagcagcaag accaccagcg tgctgcagtg ggccgagaag
      481 ggctactaca ccatgagcaa caacctggtg accctggaga acggcaagca gctgaccgtg
      541 aagagacagg gcctgtacta catctacgcc caggtgacct tctgcagcaa cagagaggcc
      601 agcagccagg cccccttcat cgccagcctg tgcctgaaga gccccggcag attcgagaga
      661 atcctgctga gagccgccaa cacccacagc agcgccaagc cctgcggcca gcagagcatc
      721 cacctgggcg gcgtgttcga gctgcagccc ggcgccagcg tgttcgtgaa cgtgaccgac
      781 cccagccagg tgagccacgg caccggcttc accagcttcg gcctgctgaa gctgggatcc
      841 gccaccaact tcagcctgct agattagccg actttcctgg gagactcagt ctatctttga
      901 ctagggataa cagggtaata tggcgcgata aaaagggtcc cgctgaggtg tcaatcgtcg
      961 gagccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1021 ttttgcatgg tcatagctgt ttcctgagag cttggcaggt gtcgcgtatg catcgatcac
     1081 gtgtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact cggatcgatc
     1141 cggcgcgcac cgtgggaaaa actccaggta gaggtacaca cgcggatagc caattcagag
     1201 taataaactg tgataatcaa ccctcatcaa tgatgacgaa ctaacccccg atatcaggtc
     1261 acatgacgaa gggaaagaga aggaaatcaa ctgtgacaaa ctgccctcaa atttggcttc
     1321 cttaaaaatt acagttcaaa aagtatgaga aaatccatgc aggctgaagg aaacagcaaa
     1381 actgtgacaa attaccctca gtaggtcaga acaaatgtga cgaaccaccc tcaaatctgt
     1441 gacagataac cctcagacta tcctgtcgtc atggaagtga tatcgcggaa ggaaaatacg
     1501 atatgagtcg tctggcggcc tttctttttc tcaatgtatg agaggcgcat tggagttctg
     1561 ctgttgatct cattaacaca gacttgcagg aagcggcggc ggaagtcagg catacgctgg
     1621 taactttgag gcagctggta acgctctatg atccagtcga ttttcagaga cacgatgcct
     1681 gagccatccg gcttacgata ctgacacagg gattcgtata aacgcatggc atacggattg
     1741 gtgatttctt ttgtttcact aagccgaaac tgcgtaaacc ggttctgtaa cccgataaag
     1801 aagggaatga gatatgggtt gatatgtaca ctgtaaagcc ctctggatgg actgtgcgca
     1861 cgtttgataa accaaggaaa agattcatag cctttttcat caccggcatc ctcttcaggg
     1921 cgataaaaaa ccacttcctt ccccgcgaaa ctcttcaatg cctgccgtat atccttactg
     1981 gcttccgcag aggtcaatcc gaatatttca gcatatttag caacatggat ctcgcagata
     2041 ccgtcatgtt cctgtagggt gccatcagat tttctgatct ggtcaacgaa cagatacagc
     2101 atacgttttt gatcccggga gagactatat gccgcctcag tgaggtcgtt tgactggacg
     2161 attcgcgggc tatttttacg tttcttgtga ttgataaccg ctgtttccgc catgacagat
     2221 ccatgtgaag tgtgacaagt ttttagattg tcacactaaa taaaaaagag tcaataagca
     2281 gggataactt tgtgaaaaaa cagcttcttc tgagggcaat ttgtcacagg gttaagggca
     2341 atttgtcaca gacaggactg tcatttgagg gtgatttgtc acactgaaag ggcaatttgt
     2401 cacaacacct tctgtagaac cagcatggat aaaggcctac aaggcgctct aaaaaagaag
     2461 atctaaaaac tataaaaaaa ataattataa aaatatcccc gtggataagt ggataacccc
     2521 aagggaagtt ttttcaggca tcgtgtgtaa gcagaatata taagtgctgt tccctggtgc
     2581 ttcctcgctc actcgaaatt cccggggata gctttatgct tgtaaaccgt tttgtgaaaa
     2641 aatttttaaa ataaaaaagg ggacctctag ggtccccaat taattagtaa tataatctat
     2701 taaaggtcat tcaaaaggtc atccaccgga tcaattcccc tgctcgcgca ggctgggtgc
     2761 caagctctcg ggtaacatca aggcccgatc cttggagccc ttgccctccc gcacgatgat
     2821 cgtgccgtga tcgaaatcca gatccttgac ccgcagttgc aaaccctcac tgatccgatt
     2881 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     2941 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     3001 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     3061 gattacgcca agcgcggtac ctgtcgcggc aacgcgctaa cagacgtagt aagaaccacc
     3121 agcattgtaa tgctggctaa agtcactttc ctgagctgta taacgatgag cgattttact
     3181 ttttctggct atgaattggc ctgctttgta acacactccg gtctatcccg tagcgccggg
     3241 catatcctgt cgcaatgtgc aaatctcgcg gcaacaacca gtgaatactt cattcacaag
     3301 cctcaccgcc tgatcgcggc agaaactggt tatagccaat caaccgtcgt tcgtgcattc
     3361 cgtgaagctg taaacaaagg tatcctgtct gtagagattg ttatcggcga tcaccgtgaa
     3421 cgtcgcgcta acctgtaccg gtttacacca tcctttttgg ccttcgcaca acaagccaaa
     3481 aatgcgctga tagaaagcaa attaaagatc tcttcagcgg caaccaaggt taaagctgtt
     3541 ctcgctaaga cattggcttt atttaatttt ttatccacac ccccatgtca aaatgatacc
     3601 ccctccccct gtcaggatga cgtggcaata aagaataaga agtcacaagt taaaaaaaca
     3661 aaaagatcag tttccggcgg tgccggaaca accagcctca aaaaattgac ttcatggatc
     3721 gctaaggcaa aagcaaaggc tgacaatctg cggttatcca aaaaacgcac tcaaaaacat
     3781 gagttcaagc agaaagtaga ggcggctgcg cggaaatatg cttacctgaa gaacaagcgt
     3841 tcgcctgata ttggcgggat atcaaacttc gataacctac cgcattgcat gacggtaaac
     3901 gaagctctta atgcggtttt agccaaaaat aaagataacg aacaatgggg tataccggca
     3961 ggattcagag ggtaatgaat tgctctaatt ataaccatgc atactttcaa cacctctagt
     4021 ttgccatgag gcaaactcat aggtgtcctg gtaagaggac actgttgcca aaactggacg
     4081 ccccattatt gcaattaata aacaactaac ggacaattct acctaacaat aagtggagtt
     4141 gcggccggcc gcgatccgct agcaaagcca cgttgtgtct caaaatctct gatgttacat
     4201 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
     4261 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctcgcgcc cgcgattaaa
     4321 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     4381 aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     4441 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     4501 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     4561 actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc
     4621 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     4681 ttgtaattgt ccttttaaca gcgatcgcgt atttcgcctc gctcaggcgc aatcacgaat
     4741 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     4801 acaagtctgg aaagaaatgc ataaactgtt gccattctca ccggattcag tcgtcactca
     4861 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     4921 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     4981 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     5041 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     5101 taattggttg taacactggc gagctctgcc acctgacgtc taagaaacca ttattatcat
     5161 gacattaacc tataaaaata ggcgtatcac gaggcagaat ttcagataaa aaaaatcctt
     5221 agctttcgct aaggatgatt tctgcacctg caccagtcag taaaacgacg gccagtagtc
     5281 aaaagcctcc gaccggaggc ttttgacttg gttcaggtgg agtgggag
//
