LOCUS       sequence-302973-        7999 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to Fc(DAPA)-Avi-6xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7999)
  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 7999)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2020.07"; journalName: "Cell"; date: "2020-08-20- 
            20"; volume: "182"; issue: "4"; pages: "1027-1043.e17"
COMMENT     Sequence Label: pD649-HAsp-SEMA6A-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             5341..5385
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5386..5403
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5543..5560)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5549..5660
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5778..5798)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5783..6112
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5963..6098
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6025..6044
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6160..6207
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6226..6825
                     /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(6226..6245)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6722..6742
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6955..7076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6992..7011)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7046..7065
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7129..7920,7921..7989))
                     /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     7752..7771
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7990..7999,1..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 cggccgccgg atttcctgag
     2761 gatagcgagc ctatcagcat cagccacggc aactacacca agcagtaccc cgtgttcgtg
     2821 ggccacaagc ctggcagaaa caccacacag agacaccggc tggacatcca gatgatcatg
     2881 attatgaacg gcaccctgta tatcgccgcc agggaccaca tctacaccgt ggacatcgat
     2941 accagccaca ccgaggaaat ctactgcagc aagaagctga cctggaagtc cagacaggcc
     3001 gacgtggaca cctgtcggat gaagggcaag cacaaggacg agtgccacaa cttcatcaag
     3061 gtgctgctga agaagaacga cgacgccctg ttcgtgtgcg gcaccaacgc cttcaatccc
     3121 agctgccgga actacaagat ggacaccctg gaacctttcg gcgacgagtt cagcggcatg
     3181 gccagatgtc cttacgatgc caagcacgcc aacgtggccc tgtttgccga tggcaagctg
     3241 tactctgcta ccgtgaccga cttcctggcc atcgacgccg tgatctacag aagcctgggc
     3301 gagagcccta cactgagaac cgtgaagcac gacagcaagt ggctgaaaga accctacttt
     3361 gtgcaggccg tggactacgg cgactacatc tactttttct tccgggaaat cgccgtcgag
     3421 tacaacacca tgggcaaagt ggtgttcccc agagtggccc aagtgtgcaa gaacgatatg
     3481 ggcggctctc agcgcgtgct ggaaaagcag tggaccagct tcctgaaggc ccggctgaat
     3541 tgctctgtgc ctggcgacag ccacttctac ttcaacatcc tgcaggctgt gaccgacgtg
     3601 atcagaatca acggcagaga tgtggtgctg gccaccttca gcacccctta caatagcatc
     3661 cctggcagcg ccgtgtgcgc ctacgatatg ctggatatcg ccagcgtgtt caccggcaga
     3721 ttcaaagagc agaagtcccc tgacagcacc tggacaccag tgcctgatga gagagtgccc
     3781 aagcctagac ctggctgttg tgccggatct agcagcctgg aaagatacgc caccagcaac
     3841 gagttccccg acgacaccct gaatttcatc aagacacacc ctctgatgga cgaggccgtg
     3901 cctagcatct tcaacagacc ctggttcctg agaaccatgg tccgataccg gctgaccaag
     3961 atcgccgtgg atacagccgc tggaccctac cagaatcaca ccgtggtctt tctgggcagc
     4021 gagaagggaa tcatcctgaa gtttctggcc cggatcggca acagcggctt tctgaacgat
     4081 agcctgttcc tggaagagat gagcgtgtac aactccgaga agtgcagcta cgacggcgtg
     4141 gaagataagc ggatcatggg catgcagctg gacagagcca gcagctctct gtacgtggcc
     4201 ttctccacct gtgtgattaa ggtgcccctg ggcagatgcg agagatacgg caagtgcaag
     4261 aaaacctgca ttgccagcag ggacccctac tgcggctgga tcaaagaagg cggagcctgc
     4321 tctcatctga gccccaatag cagactgacc ttcgagcagg acatcgagag aggcaatacc
     4381 gacggcctgg gagactgcca caattccttt gtggccctga acggccactc aagcagcctg
     4441 ctgcctagca ccacaaccag cgatagcaca gcccaagagg gctacgaaag cagaggcggc
     4501 atgctggact ggaagcacct cctggatagc cccgatagca ccgatcctct gggagccgtg
     4561 tctagccaca accaccagga caagaagggc gtcatcagag agagctacct gaagggccac
     4621 gatcagctgg tgcctgtgac aggcgcgccg ggcggcgaca agacccacac ctgccccccc
     4681 tgcccagctc cagaactgct gggagggcct tccgtgttcc tgttcccccc caagcccaag
     4741 gacaccctga tgatcagcag gacccccgag gtgacctgcg tggtggtggc cgtgtcccac
     4801 gaggacccag aggtgaagtt caactggtac gtggacggcg tggaggtgca caacgccaag
     4861 accaagccca gagaggagca gtacaacagc acctacaggg tggtgtccgt gctgaccgtg
     4921 ctgcaccagg actggctgaa cggcaaagaa tacaagtgca aagtctccaa caaggccctg
     4981 gctgccccaa tcgaaaagac aatcagcaag gccaagggcc agccacggga gccccaggtg
     5041 tacaccctgc cccccagccg ggaggagatg accaagaacc aggtgtccct gacctgtctg
     5101 gtgaagggct tctaccccag cgatatcgcc gtggagtggg agagcaacgg ccagcccgag
     5161 aacaactaca agaccacccc cccagtgctg gacagcgacg gcagcttctt cctgtacagc
     5221 aagctgaccg tggacaagtc caggtggcag cagggcaacg tgttcagctg cagcgtgatg
     5281 cacgaggccc tgcacaacca ctacacccag aagtccctga gcctgagccc cggcggcagc
     5341 ggtcttaatg atattttcga ggctcagaag attgagtggc atgaacacca ccaccatcac
     5401 cactaaggcc ggcctgtagt cactggcaca acgcgttacc cggtaagcca atcgggtata
     5461 cacggtcgtc atactgcaga cagggttctt ctactttgca agatagtctt gagtagtaaa
     5521 ataaatagat agagaaaatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     5581 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     5641 taaaatgagg aaattgcatc acaacactca accctatctc ggtctattct tttgatttat
     5701 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5761 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     5821 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     5881 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     5941 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     6001 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     6061 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     6121 gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca
     6181 tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatgac cgagtacaag
     6241 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     6301 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     6361 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     6421 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     6481 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     6541 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     6601 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     6661 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac atccgcgccc
     6721 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     6781 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacacgt gctacgagat
     6841 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     6901 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     6961 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     7021 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     7081 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcatt accaatgctt
     7141 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     7201 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gcgctgcgat
     7261 gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc agccagccgg
     7321 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7381 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7441 cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7501 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7561 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7621 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7681 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7741 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7801 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7861 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7921 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7981 aatactcata ttcttcctt
//
