LOCUS       sequence-303545-        7810 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to COMP5AP-AviTag-9xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7810)
  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 7810)
  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-ESAM-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..7810
                     /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             3460..3483
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5143..5187
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5188..5214
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(5354..5371)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5360..5471
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5589..5609)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5594..5923
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5774..5909
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5836..5855
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5971..6018
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6037..6636
                     /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(6037..6056)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6533..6553
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6766..6887
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6803..6822)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6857..6876
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6940..7731,7732..7800))
                     /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     7563..7582
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7801..7810,1..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 actgcaactg
     2761 catctgcctg ctaatagact gcaggccgtg gaaggcggag aagttgtgct gcctgcctgg
     2821 tatacactgc acggcgaagt gtctagcagc cagccttggg aagtgccctt cgtgatgtgg
     2881 ttcttcaagc agaaagagaa agaggaccag gtcctgagct acatcaacgg cgtgacaaca
     2941 agcaagcctg gcgtgtccct ggtgtacagc atgcccagca gaaacctgag cctgagactg
     3001 gaaggcctgc aagagaagga tagcggccct tacagctgca gcgtgaacgt gcaggataag
     3061 cagggcaaga gcagaggcca cagcatcaag accctggaac tgaacgtgct ggtgcctcct
     3121 gctcctccat cttgtagact gcaaggcgtg ccacacgtgg gcgccaatgt tacactgagc
     3181 tgtcagagcc ctagaagcaa gcccgctgtg cagtaccagt gggatagaca gctgcctagc
     3241 ttccagacct tcttcgcccc tgctctggat gtgatcagag gcagcctgtc tctgaccaac
     3301 ctgtctagct ctatggccgg cgtgtacgtg tgcaaggccc acaatgaagt gggcacagcc
     3361 cagtgcaacg tgacactgga agtttctaca ggccctggcg ctgctggcgc gccggaagat
     3421 caagttgatc caagattgat tgatggcaag ggcggatctc tggaggtgct gttccaggga
     3481 ccaggcggaa gcggcaatcc cggcggcgat ctggcccccc agatgctgcg cgagctgcag
     3541 gagaccaatg ccgccctgca ggacgtgcgc gagctgctgc gccaccgcgt gaaggagatc
     3601 accttcctga agaacaccgt gatggagtgc gacgcctgcg gtatgcagcc ctctagaggc
     3661 tccggctcca tcatcccagt tgaggaggag aacccggact tctggaaccg cgaggcagcc
     3721 gaggccctgg gtgccgccaa gaagctgcag cctgcacaga cagccgccaa gaacctcatc
     3781 atcttcctgg gcgatgggat gggggtgtct acggtgacag ctgccaggat cctaaaaggg
     3841 cagaagaagg acaaactggg gcctgagata cccctggcca tggaccgctt cccatatgtg
     3901 gctctgtcca agacatacaa tgtagacaaa catgtgccag acagtggagc cacagccacg
     3961 gcctacctgt gcggggtcaa gggcaacttc cagaccattg gcttgagtgc agccgcccgc
     4021 tttaaccagt gcaacacgac acgcggcaac gaggtcatct ccgtgatgaa tcgggccaag
     4081 aaagcaggga agtcagtggg agtggtaacc accacacgag tgcagcacgc ctcgccagcc
     4141 ggcacctacg cccacacggt gaaccgcaac tggtactcgg acgccgacgt gcctgcctcg
     4201 gcccgccagg aggggtgcca ggacatcgct acgcagctca tctccaacat ggacattgac
     4261 gtgatcctag gtggaggccg aaagtacatg tttcgcatgg gaaccccaga ccctgagtac
     4321 ccagatgact acagccaagg tgggaccagg ctggacggga agaatctggt gcaggaatgg
     4381 ctggcgaagc gccagggtgc ccggtatgtg tggaaccgca ctgagctcat gcaggcttcc
     4441 ctggacccgt ctgtgaccca tctcatgggt ctctttgagc ctggagacat gaaatacgag
     4501 atccaccgag actccacact ggacccctcc ctgatggaga tgacagaggc tgccctgcgc
     4561 ctgctgagca ggaacccccg cggcttcttc ctcttcgtgg agggtggtcg catcgaccat
     4621 ggtcatcatg aaagcagggc ttaccgggca ctgactgaga cgatcatgtt cgacgacgcc
     4681 attgagaggg cgggccagct caccagcgag gaggacacgc tgagcctcgt cactgccgac
     4741 cactcccacg tcttctcctt cggaggctac cccctgcgag ggagctccat cttcgggctg
     4801 gcccctggca aggcccggga caggaaggcc tacacggtcc tcctatacgg aaacggtcca
     4861 ggctatgtgc tcaaggacgg cgcccggccg gatgttaccg agagcgagag cgggagcccc
     4921 gagtatcggc agcagtcagc agtgcccctg gacgaagaga cccacgcagg cgaggacgtg
     4981 gcggtgttcg cgcgcggccc gcaggcgcac ctggttcacg gcgtgcagga gcagaccttc
     5041 atagcgcacg tcatggcctt cgccgcctgc ctggagccct acaccgcctg cgacctggcg
     5101 ccccccgccg gcaccaccga cgccgcgcac ccgggtggcg gaggactgaa cgacatcttc
     5161 gaggctcaga aaatcgaatg gcacgaacat caccatcacc atcaccatca ccattaaggc
     5221 cggcctgtag tcactggcac aacgcgttac ccggtaagcc aatcgggtat acacggtcgt
     5281 catactgcag acagggttct tctactttgc aagatagtct tgagtagtaa aataaataga
     5341 tagagaaaat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     5401 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     5461 gaaattgcat cacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     5521 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5581 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     5641 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     5701 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     5761 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     5821 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     5881 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     5941 tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc
     6001 ggcatagtat aatacgacaa ggtgaggaac taaaccatga ccgagtacaa gcccacggtg
     6061 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     6121 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     6181 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     6241 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     6301 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     6361 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     6421 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     6481 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga catccgcgcc ccgcaacctc
     6541 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     6601 cgcacctggt gcatgacccg caagcccggt gcctgacacg tgctacgaga tttcgattcc
     6661 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     6721 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     6781 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     6841 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     6901 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat taccaatgct taatcagtga
     6961 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7021 gtagataact acgatacggg agggcttacc atctggcccc agcgctgcga tgataccgcg
     7081 agaaccacgc tcaccggctc cggatttatc agcaataaac cagccagccg gaagggccga
     7141 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7201 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca tcgctacagg
     7261 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7321 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7381 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7441 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7501 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7561 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7621 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7681 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7741 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7801 attcttcctt
//
