LOCUS       sequence-478926-        8244 bp    DNA     circular SYN 17-JUL-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 8244)
  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 8244)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 17, 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-SEMA4D-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8244
                     /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"
     regulatory      2743..2752
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5587..5631
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5632..5649
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5789..5806)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5795..5906
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6024..6044)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6029..6358
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6209..6344
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6271..6290
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6406..6453
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6472..7071
                     /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(6472..6491)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6968..6988
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7201..7321
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7238..7257)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(7374..8165,8166..8234))
                     /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     7997..8016
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8235..8244,1..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 cggccgccat ggcttttgcc
     2761 cctattccta gaatcacctg ggagcaccgg gaagtgcacc tggtgcagtt tcacgagccc
     2821 gacatctaca actacagcgc cctgctgctg agcgaggaca aggacacact gtacatcggc
     2881 gccagagaag ccgtgttcgc cgtgaacgcc ctgaacatct ctgagaagca gcacgaggtg
     2941 tactggaagg tgtccgagga caaaaaggcc aagtgcgccg agaagggcaa gagcaagcag
     3001 accgagtgcc tgaactacat cagagtgctg cagccactga gcgccaccag cctgtatgtg
     3061 tgtggcacca atgccttcca gcctgcctgc gaccacctga acctgaccag cttcaagttc
     3121 ctgggcaaga acgaggacgg caagggcaga tgccctttcg atcctgctca cagctacacc
     3181 agcgtgatgg tggatggcga gctgtactcc ggaacctcct acaactttct gggcagcgag
     3241 cccatcatca gccggaactc ttctcacagc cctctgcgga ccgagtacgc cattccttgg
     3301 ctgaacgagc ccagcttcgt gtttgccgac gtgatcagaa agagccctga cagccctgat
     3361 ggcgaggacg acagagtgta cttcttcttc accgaggtgt ccgtggaata cgagttcgtg
     3421 ttccgggtgc tgatccccag aatcgccaga gtgtgcaagg gcgatcaagg cggcctgaga
     3481 accctgcaga agaagtggac cagctttctg aaggcccggc tgatctgcag cagacctgat
     3541 agcggcctgg tgttcaacgt gctgagggac gtgttcgtgc tgagaagccc tggcctgaag
     3601 gtgcccgtgt tctacgccct gtttacccct cagctgaaca acgtgggact gtccgccgtg
     3661 tgcgcctaca atctgtctac agccgaggaa gtgttcagcc acggcaagta catgcagagc
     3721 accaccgtgg aacagagcca caccaaatgg gtccgataca acggccccgt gcctaagcct
     3781 agacctggcg cctgtattga ctctgaagcc agagccgcca actacacctc cagcctgaat
     3841 ctgcccgaca agaccctgca gttcgtgaag gatcaccctc tgatggacga cagcgtgacc
     3901 cctatcgaca acagacccag actgatcaag aaggacgtca actacacaca gatcgtggtg
     3961 gaccggacac aggccctgga tggcacagtg tacgacgtga tgttcgtgtc caccgacaga
     4021 ggcgccctgc acaaggccat ttctctggaa cacgccgtgc acatcatcga ggaaacccag
     4081 ctgttccagg acttcgagcc tgtgcagaca ctgctgctgt ccagcaagaa aggcaacaga
     4141 ttcgtgtacg ccggcagcaa ctctggcgtg gtgcaagctc ctctggcctt ctgtggaaag
     4201 cacggcacat gcgaggattg cgtgctggcc agagatccct actgtgcttg gagccctcct
     4261 accgccacat gtgttgccct gcaccagaca gagagcccta gcagaggact gatccaagag
     4321 atgagcggcg acgccagcgt gtgccctgat aagagcaagg gctcctaccg gcagcacttc
     4381 tttaagcacg gcggaaccgc cgagctgaag tgcagccaga aaagcaatct ggcccgggtg
     4441 ttctggaagt ttcagaacgg cgtgctgaag gccgagtctc ctaagtatgg cctgatgggc
     4501 agaaagaacc tgctgatctt caacctgtcc gagggcgaca gcggcgtgta ccaatgtctg
     4561 agcgaagaga gagtgaagaa caagaccgtg tttcaggtcg tggccaaaca cgtgctggaa
     4621 gtgaaggtgg tgcccaagcc tgtggtggcc cctacactgt ctgtggtgca gacagagggc
     4681 agcagaattg ccaccaaggt gctggtggct tctacccagg gaagcagccc tccaacacca
     4741 gctgtgcagg ctacaagcag cggagccatt acactgcctc caaagcctgc tccaaccggc
     4801 acatcttgcg agcccaagat cgtgatcaac accgtgcctc agctgcacag cgagaaaacc
     4861 atgtacctga agtccagcga caatagaggc gcgccgggcg gcgacaagac ccacacctgc
     4921 cccccctgcc cagctccaga actgctggga gggccttccg tgttcctgtt cccccccaag
     4981 cccaaggaca ccctgatgat cagcaggacc cccgaggtga cctgcgtggt ggtggccgtg
     5041 tcccacgagg acccagaggt gaagttcaac tggtacgtgg acggcgtgga ggtgcacaac
     5101 gccaagacca agcccagaga ggagcagtac aacagcacct acagggtggt gtccgtgctg
     5161 accgtgctgc accaggactg gctgaacggc aaagaataca agtgcaaagt ctccaacaag
     5221 gccctggctg ccccaatcga aaagacaatc agcaaggcca agggccagcc acgggagccc
     5281 caggtgtaca ccctgccccc cagccgggag gagatgacca agaaccaggt gtccctgacc
     5341 tgtctggtga agggcttcta ccccagcgat atcgccgtgg agtgggagag caacggccag
     5401 cccgagaaca actacaagac caccccccca gtgctggaca gcgacggcag cttcttcctg
     5461 tacagcaagc tgaccgtgga caagtccagg tggcagcagg gcaacgtgtt cagctgcagc
     5521 gtgatgcacg aggccctgca caaccactac acccagaagt ccctgagcct gagccccggc
     5581 ggcagcggtc ttaatgatat tttcgaggct cagaagattg agtggcatga acaccaccac
     5641 catcaccact aaggccggcc tgtagtcact ggcacaacgc gttacccggt aagccaatcg
     5701 ggtatacacg gtcgtcatac tgcagacagg gttcttctac tttgcaagat agtcttgagt
     5761 agtaaaataa atagatagag aaaatcagcc tcgactgtgc cttctagttg ccagccatct
     5821 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5881 tcctaataaa atgaggaaat tgcatcacaa cactcaaccc tatctcggtc tattcttttg
     5941 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     6001 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     6061 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     6121 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     6181 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     6241 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     6301 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     6361 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
     6421 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catgaccgag
     6481 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     6541 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     6601 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     6661 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     6721 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     6781 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     6841 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     6901 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacatcc
     6961 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     7021 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acacgtgcta
     7081 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     7141 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     7201 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     7261 aataaagcat ttttttcact gcattctagt ttggtttgtc caaactcatc aatgtatctt
     7321 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcattaccaa
     7381 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     7441 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagcgct
     7501 gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat aaaccagcca
     7561 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     7621 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     7681 gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     7741 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     7801 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7861 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7921 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7981 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     8041 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     8101 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     8161 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     8221 tgttgaatac tcatattctt cctt
//
