LOCUS       Exported                5724 bp DNA     circular SYN 02-JUL-2021
DEFINITION  BBF10K_000532 in pOpen_v4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5724)
  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..5724
                     /organism="BBF10K_000532 in pOpen_v4"
     primer_bind     21..51
                     /label=Gibson-for
     CDS             52..1230
                     /codon_start=1
                     /product="Cytotoxic and regulatory T cell molecule"
                     /label=CRTAM
                     /translation="MWWRVLSLLAWFPLQEASLTNHTETITVEEGQTLTLKCVTSLRKN
                     SSLQWLTPSGFTIFLNEYPALKNSKYQLLHHSANQLSITVPNVTLQDEGVYKCLHYSDS
                     VSTKEVKVIVLATPFKPILEASVIRKQNGEEHVVLMCSTMRSKPPPQITWLLGNSMEVS
                     GGTLHEFETDGKKCNTTSTLIIHTYGKNSTVDCIIRHRGLQGRKLVAPFRFEDLVTDEE
                     TASDALERNSLSSQDPQQPTSTVSVTEDSSTSEIDKEEKEQTTQDPDLTTEANPQYLGL
                     ARKKSGILLLTLVSFLIFILFIIVQLFIMKLRKAHVIWKKENEVSEHTLESYRSRSNNE
                     ETSSEEKNGQSSHPMRCMNYITKLYSEAKTKRKENVQHSKLEEKHIQVPESIV"
     primer_bind     complement(1231..1255)
                     /label=Gibson-rev
     terminator      1374..1421
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1426..1442)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1601..1851
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(1854..2609)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      2700..2919
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     protein_bind    3339..3360
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3375..3405
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3413..3429
                     /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     3437..3453
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3526..4371
                     /codon_start=1
                     /label=repL
                     /translation="MLAKVTFLSCITMSDFTFSGYELACFVTHSGLSRSAGHILSQCAN
                     LAATTSEYFIHKPHRLIAAETGYSQSTVVRAFREAVNKGILSVEIVIGDHRERRANLYR
                     FTPSFLAFAQQAKNALIESKLKISSAATKVKAVLAKTLALFNFLSTPPCQNDTPSPCQD
                     DVAIKNKKSQVKKTKRSVSGGAGTTSLKKLTSWIAKAKAKADNLRLSKKRTQKHEFKQK
                     VEAAARKYAYLKNKRSPDIGGISNFDNLPHCMTVNEALNAVLAKNKDNEQWGIPAGFRG
                     "
     misc_feature    4479..4630
                     /label=nnpp promoters
     CDS             4670..5485
                     /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(5595..5638)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     5656..5672
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      5673..5704
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 aagaattact gacccctcgg cagactgagt cgcccggggg gatccgccac catgtggtgg
       61 cgagttctct ccttgttggc atggtttcca ttgcaagaag cgagtcttac taatcatacg
      121 gaaacaataa ctgtcgaaga aggtcaaaca ctcacactta aatgtgtcac ttcattgagg
      181 aagaattcat ccttgcaatg gctcactcca tccgggttta cgatatttct caatgaatat
      241 cctgcgctca agaatagtaa atatcaattg ctccatcatt ctgcgaatca actcagtatt
      301 actgtcccta atgttactct ccaagatgaa ggggtgtata aatgtcttca ttattccgat
      361 tccgttagta ctaaagaagt caaagttatt gtacttgcga ctcctttcaa acctattctt
      421 gaagcatccg ttattcgcaa acaaaatggg gaagaacatg ttgtacttat gtgttccacg
      481 atgcgtagta aaccaccacc tcaaataaca tggctcctcg ggaattcaat ggaagtatca
      541 ggcggaactc tccatgaatt tgaaacagat gggaagaagt gtaatacgac atcaacactc
      601 attattcata cttatggtaa gaattccacg gtagactgta taataaggca tcgaggtctc
      661 caaggtagga aactcgttgc accttttcgc tttgaagatt tggttactga tgaagaaacg
      721 gcttctgatg cattggaacg caattccctt tcttctcaag atccgcaaca accaacaagt
      781 acggtcagtg tcacggaaga ttcttccact tctgaaattg ataaagaaga gaaagaacaa
      841 actactcaag atccagattt gacaactgaa gcaaatcctc aatatttggg gcttgcacgc
      901 aagaagagtg gtattttgct cctcacactc gtttcatttc tcattttcat tctgtttatt
      961 atagtgcaac ttttcattat gaaactccgg aaggcgcatg tcatatggaa gaaagaaaac
     1021 gaagtctccg aacatacgct cgaatcatat cgatcccgtt ccaataatga agaaacgtcc
     1081 tctgaagaga agaatgggca atcatctcat cctatgaggt gtatgaatta cattacaaaa
     1141 ttgtactccg aagcaaagac taaacgcaag gaaaatgttc aacattccaa actcgaagag
     1201 aaacatattc aagttccaga gagcatcgtg ggatccgcca ccaacttcag cctgctagta
     1261 ttcttacagc cagccgaatg ggaaagccta ggtcatctag ggataacagg gtaatgataa
     1321 gatgatatgc tgctggcgct gaggtgtcaa tcgtcggagc cgctgagcaa taactagcat
     1381 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gcatggtcat agctgtttcc
     1441 tgagagcttg gcaggtgtcg cgtatgcatc gatcacgtgt ccacgttctt taatagtgga
     1501 ctcttgttcc aaactggaac aacactcgga tcgatccggc gcgcaccgtg ggaaaaactc
     1561 caggtagagg tacacacgcg gatagccaat tcagagtaat aaactgtgat aatcaaccct
     1621 catcaatgat gacgaactaa cccccgatat caggtcacat gacgaaggga aagagaagga
     1681 aatcaactgt gacaaactgc cctcaaattt ggcttcctta aaaattacag ttcaaaaagt
     1741 atgagaaaat ccatgcaggc tgaaggaaac agcaaaactg tgacaaatta ccctcagtag
     1801 gtcagaacaa atgtgacgaa ccaccctcaa atctgtgaca gataaccctc agactatcct
     1861 gtcgtcatgg aagtgatatc gcggaaggaa aatacgatat gagtcgtctg gcggcctttc
     1921 tttttctcaa tgtatgagag gcgcattgga gttctgctgt tgatctcatt aacacagact
     1981 tgcaggaagc ggcggcggaa gtcaggcata cgctggtaac tttgaggcag ctggtaacgc
     2041 tctatgatcc agtcgatttt cagagacacg atgcctgagc catccggctt acgatactga
     2101 cacagggatt cgtataaacg catggcatac ggattggtga tttcttttgt ttcactaagc
     2161 cgaaactgcg taaaccggtt ctgtaacccg ataaagaagg gaatgagata tgggttgata
     2221 tgtacactgt aaagccctct ggatggactg tgcgcacgtt tgataaacca aggaaaagat
     2281 tcatagcctt tttcatcacc ggcatcctct tcagggcgat aaaaaaccac ttccttcccc
     2341 gcgaaactct tcaatgcctg ccgtatatcc ttactggctt ccgcagaggt caatccgaat
     2401 atttcagcat atttagcaac atggatctcg cagataccgt catgttcctg tagggtgcca
     2461 tcagattttc tgatctggtc aacgaacaga tacagcatac gtttttgatc ccgggagaga
     2521 ctatatgccg cctcagtgag gtcgtttgac tggacgattc gcgggctatt tttacgtttc
     2581 ttgtgattga taaccgctgt ttccgccatg acagatccat gtgaagtgtg acaagttttt
     2641 agattgtcac actaaataaa aaagagtcaa taagcaggga taactttgtg aaaaaacagc
     2701 ttcttctgag ggcaatttgt cacagggtta agggcaattt gtcacagaca ggactgtcat
     2761 ttgagggtga tttgtcacac tgaaagggca atttgtcaca acaccttctg tagaaccagc
     2821 atggataaag gcctacaagg cgctctaaaa aagaagatct aaaaactata aaaaaaataa
     2881 ttataaaaat atccccgtgg ataagtggat aaccccaagg gaagtttttt caggcatcgt
     2941 gtgtaagcag aatatataag tgctgttccc tggtgcttcc tcgctcactc gaaattcccg
     3001 gggatagctt tatgcttgta aaccgttttg tgaaaaaatt tttaaaataa aaaaggggac
     3061 ctctagggtc cccaattaat tagtaatata atctattaaa ggtcattcaa aaggtcatcc
     3121 accggatcaa ttcccctgct cgcgcaggct gggtgccaag ctctcgggta acatcaaggc
     3181 ccgatccttg gagcccttgc cctcccgcac gatgatcgtg ccgtgatcga aatccagatc
     3241 cttgacccgc agttgcaaac cctcactgat ccgattcatt aatgcagctg gcacgacagg
     3301 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     3361 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     3421 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cggtacctgt
     3481 cgcggcaacg cgctaacaga cgtagtaaga accaccagca ttgtaatgct ggctaaagtc
     3541 actttcctga gctgtataac gatgagcgat tttacttttt ctggctatga attggcctgc
     3601 tttgtaacac actccggtct atcccgtagc gccgggcata tcctgtcgca atgtgcaaat
     3661 ctcgcggcaa caaccagtga atacttcatt cacaagcctc accgcctgat cgcggcagaa
     3721 actggttata gccaatcaac cgtcgttcgt gcattccgtg aagctgtaaa caaaggtatc
     3781 ctgtctgtag agattgttat cggcgatcac cgtgaacgtc gcgctaacct gtaccggttt
     3841 acaccatcct ttttggcctt cgcacaacaa gccaaaaatg cgctgataga aagcaaatta
     3901 aagatctctt cagcggcaac caaggttaaa gctgttctcg ctaagacatt ggctttattt
     3961 aattttttat ccacaccccc atgtcaaaat gataccccct ccccctgtca ggatgacgtg
     4021 gcaataaaga ataagaagtc acaagttaaa aaaacaaaaa gatcagtttc cggcggtgcc
     4081 ggaacaacca gcctcaaaaa attgacttca tggatcgcta aggcaaaagc aaaggctgac
     4141 aatctgcggt tatccaaaaa acgcactcaa aaacatgagt tcaagcagaa agtagaggcg
     4201 gctgcgcgga aatatgctta cctgaagaac aagcgttcgc ctgatattgg cgggatatca
     4261 aacttcgata acctaccgca ttgcatgacg gtaaacgaag ctcttaatgc ggttttagcc
     4321 aaaaataaag ataacgaaca atggggtata ccggcaggat tcagagggta atgaattgct
     4381 ctaattataa ccatgcatac tttcaacacc tctagtttgc catgaggcaa actcataggt
     4441 gtcctggtaa gaggacactg ttgccaaaac tggacgcccc attattgcaa ttaataaaca
     4501 actaacggac aattctacct aacaataagt ggagttgcgg ccggccgcga tccgctagca
     4561 aagccacgtt gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc
     4621 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     4681 tcaacgggaa acgtcttgct cgcgcccgcg attaaattcc aacatggatg ctgatttata
     4741 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta
     4801 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     4861 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     4921 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa
     4981 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     5041 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     5101 tcgcgtattt cgcctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     5161 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     5221 actgttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     5281 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     5341 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     5401 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     5461 tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcgagc
     5521 tctgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     5581 tatcacgagg cagaatttca gataaaaaaa atccttagct ttcgctaagg atgatttctg
     5641 cacctgcacc agtcagtaaa acgacggcca gtagtcaaaa gcctccgacc ggaggctttt
     5701 gacttggttc aggtggagtg ggag
//
