LOCUS Exported 7840 bp ds-DNA circular SYN 13-JAN-2023 DEFINITION Mammalian expression of cell-surface protein extracellular domain fused to Fc(DAPA)-Avi-6xHis. Protein is secreted from cells.. ACCESSION . VERSION . KEYWORDS pD649-HAsp-SEMA6B-Fc(DAPA)-AviTag-6xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7840) AUTHORS Wojtowicz WM, Vielmetter J, Fernandes RA, Siepe DH, Eastman CL, Chisholm GB, Cox S, Klock H, Anderson PW, Rue SM, Miller JJ, Glaser SM, Bragstad ML, Vance J, Lam AW, Lesley SA, Zinn K, Garcia KC TITLE A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions. JOURNAL Cell. 2020 Aug 20;182(4):1027-1043.e17. doi: 10.1016/j.cell.2020.07.025. PUBMED 32822567 REFERENCE 2 (bases 1 to 7840) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 13, 2023 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7840 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 124..143 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 149..168 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 254..842 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 743..762 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 996..1013 /label=L4440 /note="L4440 vector, forward primer" primer_bind 1111..1128 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 1112..1128 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 1584..1600 /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)." enhancer 1767..2146 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 2147..2350 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 2301..2321 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 2347..2371 /label=LNCX /note="Human CMV promoter, forward primer" promoter 2581..2599 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 2581..2598 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 2612..2631 /label=pMT2-F /note="Synthetic intron, forward primer" CDS 5182..5226 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 5227..5244 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5384..5401) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5390..5501 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5619..5639) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5624..5953 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5804..5939 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5866..5885 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 6001..6048 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 6067..6666 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(6067..6086) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6563..6583 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6796..6917 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6833..6852) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6887..6906 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(6970..7830) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7593..7612 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7831..82) /gene="bla" /label=AmpR promoter ORIGIN 1 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 61 tgtatttaga aaaataaaca aataggggtc agtgttacaa ccaattaacc aattctgaac 121 attatcgcga gcccatttat acctgaatat ggctcataac accccttgct catgaccaaa 181 atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga 241 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 301 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 361 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt 421 tagcccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 481 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 541 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 601 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 661 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 721 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 781 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 841 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 901 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 961 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 1021 aaggcgagag tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc 1081 tttttgcgtt tctacaaact ctttctgtgt tgtaaaacga cggccagtct taagctcggg 1141 ccccctgggc ggttctgata acgagtaatc gttaatccgc aaataacgta aaaacccgct 1201 tcggcgggtt tttttatggg gggagtttag ggaaagagca tttgtcagaa tatttaaggg 1261 cgcctgtcac tttgcttgat atatgagaat tatttaacct tataaatgag aaaaaagcaa 1321 cgcactttaa ataagatacg ttgctttttc gattgatgaa cacctataat taaactattc 1381 atctattatt tatgattttt tgtatataca atatttctag tttgttaaag agaattaaga 1441 aaataaatct cgaaaataat aaagggaaaa tcagtttttg atatcaaaat tatacatgtc 1501 aacgataata caaaatataa tacaaactat aagatgttat cagtatttat tatcatttag 1561 aataaatttt gtgtcgccct taattgtgag cggataacaa ttacgagctt catgcacagt 1621 ggcgttgact caatattggc cattagccat attattcatt ggttatatag cataaatcaa 1681 tattggctat tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg 1741 ctcatgtcca atatgaccgc catgttggca ttgattattg actagttatt aatagtaatc 1801 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 1861 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 1921 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 1981 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 2041 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 2101 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 2161 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 2221 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 2281 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 2341 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga 2401 cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac 2461 gcggattccc cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc 2521 cttggcttcg ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg 2581 atttaggtga cactatagaa taacatccac tttgcctttc tctccacagg tgtccactcc 2641 caggtccaac tgcacctcgg ttctatcgaa aacgcgcctg ctagcgccac catgaagacc 2701 atcattgccc tgagctacat cttctgcctg gtgttcgctg cggccgcctt gtttcctgaa 2761 gaacctcctc ctctgtctgt ggcccctcgg gactacctga atcactaccc tgtgtttgtt 2821 ggcagcggcc ctggtagact tacaccagct gaaggcgccg acgacctgaa tatccagcgg 2881 gtgctgagag tgaacagaac cctgttcatc ggcgaccggg acaacctgta cagagtggaa 2941 ctggaacctc ctaccagcac cgagctgaga taccagagaa agctgacctg gcggagcaac 3001 cccagcgaca tcaacgtgtg cagaatgaag ggcaagcaag agggcgagtg ccggaacttt 3061 gtgaaggtgc tgctgctgag agatgagagc acactgttcg tgtgcggcag caacgccttc 3121 aatcccgtgt gcgccaacta cagcatcgac acactgcagc ccgtgggcga caatatctct 3181 ggcatggcca gatgtcccta cgatcccaaa catgccaacg tggccctgtt cagcgacggc 3241 atgctgttta ccgccaccgt gaccgacttt ctggccatcg atgccgtgat ctacagaagc 3301 ctgggcgaca gacctacact gagaaccgtg aagcacgaca gcaagtggtt caaagaaccc 3361 tacttcgtgc acgccgtgga atggggcagc cacgtgtact ttttcttccg ggaaatcgcc 3421 atggaattca actacctgga aaaggtggtg gtgtccagag tggccagagt gtgcaaaaac 3481 gatgtcggcg gcagccccag agtgctggaa aagcagtgga caagcttcct gaaggcccgg 3541 ctgaattgct ctgtgcctgg cgacagccac ttctacttca atgtgctgca ggccgtgacc 3601 ggcgtggtgt ctcttggagg tagacctgtg gtgctggccg tgtttagcac ccctagcaat 3661 tctatccctg gcagcgccgt gtgtgccttc gatctgacac aagtggccgc cgtgttcgag 3721 ggcagattca gagagcagaa gtcccctgag agcatctgga cccctgtgcc tgaagatcag 3781 gtgccaagac ctagacctgg ctgttgtgct gcccctggca tgcagtacaa tgcctcttct 3841 gccctgcctg acgacatcct gaacttcgtg aaaacacacc ctctgatgga cgaggccgtg 3901 ccttctcttg gacatgcccc ttggattctg cggaccctga tgagacatca gctgacaaga 3961 gtggccgtgg atgttggagc cggaccttgg ggaaatcaga ccgtggtgtt tctgggctct 4021 gaggccggca cagtgctgaa atttcttgtg cggcctaatg ccagcacaag cggaacaagc 4081 ggcctgagcg tgttcctgga agagttcgag acatacagac ccgacagatg cggtagacca 4141 ggcggcggag aaacaggaca gagactgctg tctcttgagc tggatgctgc ttctggtggc 4201 ctgctggccg cctttcctag atgtgttgtt agagtgcctg tggccaggtg ccagcagtac 4261 agcggctgca tgaagaactg catcggctct caggaccctt actgtggatg ggcccctgac 4321 ggcagctgta tctttctgtc tcctggcacc agagccgcct tcgaacaaga tgtgtctggc 4381 gcctctacaa gcggactggg agattgcaca ggactgctga gggccagcct gtctgaagat 4441 agagctggcc tggtgtccgt gaacctgctg gtcacatctt ctggcgcgcc gggcggcgac 4501 aagacccaca cctgcccccc ctgcccagct ccagaactgc tgggagggcc ttccgtgttc 4561 ctgttccccc ccaagcccaa ggacaccctg atgatcagca ggacccccga ggtgacctgc 4621 gtggtggtgg ccgtgtccca cgaggaccca gaggtgaagt tcaactggta cgtggacggc 4681 gtggaggtgc acaacgccaa gaccaagccc agagaggagc agtacaacag cacctacagg 4741 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaaaga atacaagtgc 4801 aaagtctcca acaaggccct ggctgcccca atcgaaaaga caatcagcaa ggccaagggc 4861 cagccacggg agccccaggt gtacaccctg ccccccagcc gggaggagat gaccaagaac 4921 caggtgtccc tgacctgtct ggtgaagggc ttctacccca gcgatatcgc cgtggagtgg 4981 gagagcaacg gccagcccga gaacaactac aagaccaccc ccccagtgct ggacagcgac 5041 ggcagcttct tcctgtacag caagctgacc gtggacaagt ccaggtggca gcagggcaac 5101 gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 5161 agcctgagcc ccggcggcag cggtcttaat gatattttcg aggctcagaa gattgagtgg 5221 catgaacacc accaccatca ccactaaggc cggcctgtag tcactggcac aacgcgttac 5281 ccggtaagcc aatcgggtat acacggtcgt catactgcag acagggttct tctactttgc 5341 aagatagtct tgagtagtaa aataaataga tagagaaaat cagcctcgac tgtgccttct 5401 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 5461 actcccactg tcctttccta ataaaatgag gaaattgcat cacaacactc aaccctatct 5521 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 5581 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg 5641 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 5701 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 5761 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc 5821 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 5881 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 5941 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt 6001 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac 6061 taaaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg 6121 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat 6181 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg 6241 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg 6301 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg 6361 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc 6421 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt 6481 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc 6541 ttcctggaga catccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc 6601 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt 6661 gcctgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 6721 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 6781 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 6841 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 6901 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 6961 tcatggtcat taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 7021 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 7081 atctggcccc agcgctgcga tgataccgcg agaaccacgc tcaccggctc cggatttatc 7141 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 7201 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 7261 tttgcgcaac gttgttgcca tcgctacagg catcgtggtg tcacgctcgt cgtttggtat 7321 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 7381 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 7441 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 7501 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 7561 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 7621 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 7681 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 7741 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 7801 aagggcgaca cggaaatgtt gaatactcat attcttcctt //