LOCUS       sequence-302837-        7426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of secreted protein fused to 
            Fc(DAPA)-Avi-6xHis..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7426)
  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 7426)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 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-ROBO4-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7426
                     /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             4768..4812
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4813..4830
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4970..4987)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4976..5087
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5205..5225)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5210..5539
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5390..5525
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5452..5471
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5587..5634
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5653..6252
                     /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(5653..5672)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6149..6169
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6382..6503
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6419..6438)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6473..6492
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6556..7347,7348..7416))
                     /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     7179..7198
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7417..7426,1..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 cggccgccca agattctcca
     2761 cctcagattc tggtgcaccc tcaggaccag ctgtttcaag gacctggacc tgccagaatg
     2821 agctgtcagg cttctggaca gcctcctcca accatcagat ggctgctgaa tggccagcct
     2881 ctgagcatgg tgcctccaga tcctcaccat ctgctgcctg atggaacact gctgctgctt
     2941 cagcctcctg ccagaggaca tgctcatgat ggacaggccc tgagcaccga tctgggcgtg
     3001 tacacatgcg aggccagcaa tagactgggc acagctgtta gcagaggcgc cagactgtct
     3061 gtggccgtgc tgagagagga tttccagatc cagcctagag acatggtggc cgtcgtgggc
     3121 gagcagttta cactggaatg tggacctcca tggggacacc ctgagcctac agtgtcttgg
     3181 tggaaggatg gcaagccact ggctctgcaa cctggcagac ataccgtttc tggcggcagc
     3241 ctgctgatgg ctagagccga gaaatccgac gagggcacct atatgtgcgt ggccacaaat
     3301 agcgccggac acagagaatc cagagccgct agagtgtcca tccaagagcc tcaggattac
     3361 accgagcctg tggaactgct ggccgtgcgg attcagctgg aaaacgtgac cctgctgaac
     3421 cccgatcctg ccgagggacc taaacctaga cctgctgtgt ggctgagctg gaaggtttcc
     3481 ggacctgctg ctcctgctca gagctacaca gccctgttca gaacacagac agctccaggt
     3541 ggacaaggcg ccccttgggc tgaagaactt ctggctggat ggcagtctgc cgaactcgga
     3601 ggactgcact ggggccaaga ctacgagttt aaagtgcggc ctagcagcgg cagagctaga
     3661 ggccctgatt ctaacgtcct gctgctgaga ctgcccgaga aagtgcctag cgctcctcca
     3721 caagaagtga ccctgaagcc tggcaatggc accgtgtttg tgtcttgggt gccacctcca
     3781 gccgagaacc acaatggcat catccggggc tatcaagtgt ggtccctggg caatacttcc
     3841 ctgcctcctg ctaattggac cgttgtggga gagcagaccc agctcgagat tgccacacac
     3901 atgcctggca gctattgtgt gcaggttgcc gctgttacag gtgctggtgc tggcgaacct
     3961 agcagacctg tttgtctgct gctggaacag gccatggaaa gagccacaca agagcctagc
     4021 gagcacggcc cttggacact ggaacaactg agggccacac tgaagcgagg cgcgccgggc
     4081 ggcgacaaga cccacacctg ccccccctgc ccagctccag aactgctggg agggccttcc
     4141 gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagcaggac ccccgaggtg
     4201 acctgcgtgg tggtggccgt gtcccacgag gacccagagg tgaagttcaa ctggtacgtg
     4261 gacggcgtgg aggtgcacaa cgccaagacc aagcccagag aggagcagta caacagcacc
     4321 tacagggtgg tgtccgtgct gaccgtgctg caccaggact ggctgaacgg caaagaatac
     4381 aagtgcaaag tctccaacaa ggccctggct gccccaatcg aaaagacaat cagcaaggcc
     4441 aagggccagc cacgggagcc ccaggtgtac accctgcccc ccagccggga ggagatgacc
     4501 aagaaccagg tgtccctgac ctgtctggtg aagggcttct accccagcga tatcgccgtg
     4561 gagtgggaga gcaacggcca gcccgagaac aactacaaga ccaccccccc agtgctggac
     4621 agcgacggca gcttcttcct gtacagcaag ctgaccgtgg acaagtccag gtggcagcag
     4681 ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag
     4741 tccctgagcc tgagccccgg cggcagcggt cttaatgata ttttcgaggc tcagaagatt
     4801 gagtggcatg aacaccacca ccatcaccac taaggccggc ctgtagtcac tggcacaacg
     4861 cgttacccgg taagccaatc gggtatacac ggtcgtcata ctgcagacag ggttcttcta
     4921 ctttgcaaga tagtcttgag tagtaaaata aatagataga gaaaatcagc ctcgactgtg
     4981 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     5041 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcaca acactcaacc
     5101 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     5161 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     5221 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     5281 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     5341 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     5401 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     5461 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     5521 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     5581 gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
     5641 aggaactaaa ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     5701 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     5761 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     5821 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     5881 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     5941 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     6001 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     6061 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     6121 cccgccttcc tggagacatc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     6181 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     6241 cccggtgcct gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg
     6301 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     6361 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     6421 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     6481 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     6541 gcgtaatcat ggtcattacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6601 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6661 cttaccatct ggccccagcg ctgcgatgat accgcgagaa ccacgctcac cggctccgga
     6721 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6781 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6841 taatagtttg cgcaacgttg ttgccatcgc tacaggcatc gtggtgtcac gctcgtcgtt
     6901 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6961 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7021 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7081 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7141 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7201 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7261 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7321 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7381 gggaataagg gcgacacgga aatgttgaat actcatattc ttcctt
//
