LOCUS Exported 5391 bp DNA circular SYN 02-JUL-2021 DEFINITION BBF10K_000514 in pOpen_v4. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5391) 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..5391 /organism="BBF10K_000514 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..897 /codon_start=1 /product="TMIGD2 transmembrane and immunoglobulin domain containing 2" /label=CD28H /translation="MGSPGMVLGLLVQIWALQEASSLSVQQGPNLLQVRQGSQATLVCQ VDQATAWERLRVKWTKDGAILCQPYITNGSLSLGVCGPQGRLSWQAPSHLTLQLDPVSL NHSGAYVCWAAVEIPELEEAEGNITRLFVDPDDPTQNRNRIASFPGFLFVLLGVGSMGV AAIVWGAWFWGRRSCQQRDSGNSPGNAFYSNVLYRPRGAPKKSEDCSGEGKDQRGQSIY STSFPQPAPRQPHLASRPCPSPRPCPSPRPGHPVSMVRVSPRPSPTQQPRPKGFPKVGE E" primer_bind complement(898..922) /label=Gibson-rev terminator 1041..1088 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind complement(1093..1109) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" misc_feature 1268..1518 /gene="incC" /label=incC /note="incompatibility region of the bacterial F plasmid" CDS complement(1521..2276) /codon_start=1 /gene="repE" /product="replication initiation protein for the bacterial F plasmid" /label=repE /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM RLSYIEKKKGRQTTHIVFSFRDITSMTTG" rep_origin 2367..2586 /label=ori2 /note="secondary origin of replication for the bacterial F plasmid; also known as oriS" protein_bind 3006..3027 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3042..3072 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3080..3096 /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 3104..3120 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" CDS 3193..4038 /codon_start=1 /label=repL /translation="MLAKVTFLSCITMSDFTFSGYELACFVTHSGLSRSAGHILSQCAN LAATTSEYFIHKPHRLIAAETGYSQSTVVRAFREAVNKGILSVEIVIGDHRERRANLYR FTPSFLAFAQQAKNALIESKLKISSAATKVKAVLAKTLALFNFLSTPPCQNDTPSPCQD DVAIKNKKSQVKKTKRSVSGGAGTTSLKKLTSWIAKAKAKADNLRLSKKRTQKHEFKQK VEAAARKYAYLKNKRSPDIGGISNFDNLPHCMTVNEALNAVLAKNKDNEQWGIPAGFRG " misc_feature 4146..4297 /label=nnpp promoters CDS 4337..5152 /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(5262..5305) /label=bacterial terminator /note="putative bacterial transcription terminator" primer_bind 5323..5339 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" terminator 5340..5371 /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" ORIGIN 1 aagaattact gacccctcgg cagactgagt cgcccggggg gatccgccac catgggcagc 61 cctgggatgg ttcttggtct ccttgttcaa atttgggctc ttcaagaagc atcttccctt 121 tctgttcaac aaggacctaa tcttcttcaa gtacggcaag gttctcaagc aacacttgtt 181 tgtcaagttg atcaagctac tgcttgggaa cgactccggg tcaaatggac aaaagatgga 241 gcaattctct gtcaacctta tattactaat ggttcccttt ctctcggagt atgtgggccg 301 caaggtcgat tgtcctggca agcaccttct catctcacac tccaattgga cccagtatcc 361 cttaatcata gtggggctta tgtttgttgg gcagcggtag aaatacctga acttgaagaa 421 gcggaaggta atattacgag gcttttcgtt gatccagatg atccaacaca aaatcgcaat 481 aggatagcat catttccagg tttcttgttt gttttgctcg gagtcggttc tatgggagtc 541 gcagcaatag tttggggagc ttggttttgg ggtaggcggt cctgtcaaca acgtgattca 601 gggaattccc caggtaatgc attttattcc aatgtactct atcgaccacg tggggctccg 661 aagaaatcag aagattgttc cggggaaggt aaagatcaaa gagggcaatc catttattcc 721 acgtcctttc cacaaccagc tccacggcaa ccacatctcg cttctcggcc ttgtccatcc 781 ccacgtccat gtccttctcc tcggccagga catccagtat ccatggttag ggtttcccca 841 cggccttctc cgacgcaaca accacggcca aaaggttttc ctaaagtcgg cgaggaggga 901 tccgccacca acttcagcct gctagagttg cacctagaat ccgggagcta gcgcagctgc 961 gaagtaggga taacagggta atacatgccc gcagcctgat ccgcgctgag gtgtcaatcg 1021 tcggagccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg 1081 gttttttgca tggtcatagc tgtttcctga gagcttggca ggtgtcgcgt atgcatcgat 1141 cacgtgtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcggatcg 1201 atccggcgcg caccgtggga aaaactccag gtagaggtac acacgcggat agccaattca 1261 gagtaataaa ctgtgataat caaccctcat caatgatgac gaactaaccc ccgatatcag 1321 gtcacatgac gaagggaaag agaaggaaat caactgtgac aaactgccct caaatttggc 1381 ttccttaaaa attacagttc aaaaagtatg agaaaatcca tgcaggctga aggaaacagc 1441 aaaactgtga caaattaccc tcagtaggtc agaacaaatg tgacgaacca ccctcaaatc 1501 tgtgacagat aaccctcaga ctatcctgtc gtcatggaag tgatatcgcg gaaggaaaat 1561 acgatatgag tcgtctggcg gcctttcttt ttctcaatgt atgagaggcg cattggagtt 1621 ctgctgttga tctcattaac acagacttgc aggaagcggc ggcggaagtc aggcatacgc 1681 tggtaacttt gaggcagctg gtaacgctct atgatccagt cgattttcag agacacgatg 1741 cctgagccat ccggcttacg atactgacac agggattcgt ataaacgcat ggcatacgga 1801 ttggtgattt cttttgtttc actaagccga aactgcgtaa accggttctg taacccgata 1861 aagaagggaa tgagatatgg gttgatatgt acactgtaaa gccctctgga tggactgtgc 1921 gcacgtttga taaaccaagg aaaagattca tagccttttt catcaccggc atcctcttca 1981 gggcgataaa aaaccacttc cttccccgcg aaactcttca atgcctgccg tatatcctta 2041 ctggcttccg cagaggtcaa tccgaatatt tcagcatatt tagcaacatg gatctcgcag 2101 ataccgtcat gttcctgtag ggtgccatca gattttctga tctggtcaac gaacagatac 2161 agcatacgtt tttgatcccg ggagagacta tatgccgcct cagtgaggtc gtttgactgg 2221 acgattcgcg ggctattttt acgtttcttg tgattgataa ccgctgtttc cgccatgaca 2281 gatccatgtg aagtgtgaca agtttttaga ttgtcacact aaataaaaaa gagtcaataa 2341 gcagggataa ctttgtgaaa aaacagcttc ttctgagggc aatttgtcac agggttaagg 2401 gcaatttgtc acagacagga ctgtcatttg agggtgattt gtcacactga aagggcaatt 2461 tgtcacaaca ccttctgtag aaccagcatg gataaaggcc tacaaggcgc tctaaaaaag 2521 aagatctaaa aactataaaa aaaataatta taaaaatatc cccgtggata agtggataac 2581 cccaagggaa gttttttcag gcatcgtgtg taagcagaat atataagtgc tgttccctgg 2641 tgcttcctcg ctcactcgaa attcccgggg atagctttat gcttgtaaac cgttttgtga 2701 aaaaattttt aaaataaaaa aggggacctc tagggtcccc aattaattag taatataatc 2761 tattaaaggt cattcaaaag gtcatccacc ggatcaattc ccctgctcgc gcaggctggg 2821 tgccaagctc tcgggtaaca tcaaggcccg atccttggag cccttgccct cccgcacgat 2881 gatcgtgccg tgatcgaaat ccagatcctt gacccgcagt tgcaaaccct cactgatccg 2941 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 3001 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 3061 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 3121 catgattacg ccaagcgcgg tacctgtcgc ggcaacgcgc taacagacgt agtaagaacc 3181 accagcattg taatgctggc taaagtcact ttcctgagct gtataacgat gagcgatttt 3241 actttttctg gctatgaatt ggcctgcttt gtaacacact ccggtctatc ccgtagcgcc 3301 gggcatatcc tgtcgcaatg tgcaaatctc gcggcaacaa ccagtgaata cttcattcac 3361 aagcctcacc gcctgatcgc ggcagaaact ggttatagcc aatcaaccgt cgttcgtgca 3421 ttccgtgaag ctgtaaacaa aggtatcctg tctgtagaga ttgttatcgg cgatcaccgt 3481 gaacgtcgcg ctaacctgta ccggtttaca ccatcctttt tggccttcgc acaacaagcc 3541 aaaaatgcgc tgatagaaag caaattaaag atctcttcag cggcaaccaa ggttaaagct 3601 gttctcgcta agacattggc tttatttaat tttttatcca cacccccatg tcaaaatgat 3661 accccctccc cctgtcagga tgacgtggca ataaagaata agaagtcaca agttaaaaaa 3721 acaaaaagat cagtttccgg cggtgccgga acaaccagcc tcaaaaaatt gacttcatgg 3781 atcgctaagg caaaagcaaa ggctgacaat ctgcggttat ccaaaaaacg cactcaaaaa 3841 catgagttca agcagaaagt agaggcggct gcgcggaaat atgcttacct gaagaacaag 3901 cgttcgcctg atattggcgg gatatcaaac ttcgataacc taccgcattg catgacggta 3961 aacgaagctc ttaatgcggt tttagccaaa aataaagata acgaacaatg gggtataccg 4021 gcaggattca gagggtaatg aattgctcta attataacca tgcatacttt caacacctct 4081 agtttgccat gaggcaaact cataggtgtc ctggtaagag gacactgttg ccaaaactgg 4141 acgccccatt attgcaatta ataaacaact aacggacaat tctacctaac aataagtgga 4201 gttgcggccg gccgcgatcc gctagcaaag ccacgttgtg tctcaaaatc tctgatgtta 4261 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag 4321 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcgc gcccgcgatt 4381 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca 4441 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa 4501 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct 4561 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg 4621 gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga 4681 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc 4741 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgc ctcgctcagg cgcaatcacg 4801 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt 4861 tgaacaagtc tggaaagaaa tgcataaact gttgccattc tcaccggatt cagtcgtcac 4921 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat 4981 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg 5041 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa 5101 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt 5161 ggttaattgg ttgtaacact ggcgagctct gccacctgac gtctaagaaa ccattattat 5221 catgacatta acctataaaa ataggcgtat cacgaggcag aatttcagat aaaaaaaatc 5281 cttagctttc gctaaggatg atttctgcac ctgcaccagt cagtaaaacg acggccagta 5341 gtcaaaagcc tccgaccgga ggcttttgac ttggttcagg tggagtggga g //