LOCUS       sequence-493002-        7714 bp    DNA     circular SYN 18-NOV-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 7714)
  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 7714)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 18, 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-MCAM-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(50..67)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    56..167
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(285..305)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        290..619
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      470..605
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        667..714
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             733..1332
                     /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(733..752)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1229..1249
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1462..1583
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1499..1518)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1553..1572
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1636..2427,2428..2496))
                     /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     2259..2278
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2497..2588)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2630..2649
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2655..2674
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2760..3348
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3249..3268
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3502..3519
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3617..3634
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3618..3634
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4090..4106
                     /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        4273..4652
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4653..4856
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4807..4827
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4853..4877
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        5087..5105
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5087..5104
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     5118..5137
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             7562..7606
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             7607..7624
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 ctttgcaaga tagtcttgag tagtaaaata aatagataga gaaaatcagc ctcgactgtg
       61 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
      121 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcaca acactcaacc
      181 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
      241 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
      301 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
      361 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
      421 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
      481 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
      541 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
      601 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
      661 gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
      721 aggaactaaa ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
      781 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
      841 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
      901 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
      961 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     1021 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     1081 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     1141 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     1201 cccgccttcc tggagacatc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     1261 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     1321 cccggtgcct gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg
     1381 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     1441 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     1501 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     1561 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     1621 gcgtaatcat ggtcattacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     1681 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     1741 cttaccatct ggccccagcg ctgcgatgat accgcgagaa ccacgctcac cggctccgga
     1801 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     1861 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     1921 taatagtttg cgcaacgttg ttgccatcgc tacaggcatc gtggtgtcac gctcgtcgtt
     1981 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2041 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2101 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2161 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2221 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2281 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     2341 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     2401 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     2461 gggaataagg gcgacacgga aatgttgaat actcatattc ttcctttttc aatattattg
     2521 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     2581 taaacaaata ggggtcagtg ttacaaccaa ttaaccaatt ctgaacatta tcgcgagccc
     2641 atttatacct gaatatggct cataacaccc cttgctcatg accaaaatcc cttaacgtga
     2701 gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2761 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2821 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2881 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc ccaccacttc
     2941 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3001 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3061 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3121 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3181 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3241 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3301 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3361 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3421 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3481 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaagg cgagagtagg
     3541 gaactgccag gcatcaaact aagcagaagg cccctgacgg atggcctttt tgcgtttcta
     3601 caaactcttt ctgtgttgta aaacgacggc cagtcttaag ctcgggcccc ctgggcggtt
     3661 ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa cccgcttcgg cgggtttttt
     3721 tatgggggga gtttagggaa agagcatttg tcagaatatt taagggcgcc tgtcactttg
     3781 cttgatatat gagaattatt taaccttata aatgagaaaa aagcaacgca ctttaaataa
     3841 gatacgttgc tttttcgatt gatgaacacc tataattaaa ctattcatct attatttatg
     3901 attttttgta tatacaatat ttctagtttg ttaaagagaa ttaagaaaat aaatctcgaa
     3961 aataataaag ggaaaatcag tttttgatat caaaattata catgtcaacg ataatacaaa
     4021 atataataca aactataaga tgttatcagt atttattatc atttagaata aattttgtgt
     4081 cgcccttaat tgtgagcgga taacaattac gagcttcatg cacagtggcg ttgactcaat
     4141 attggccatt agccatatta ttcattggtt atatagcata aatcaatatt ggctattggc
     4201 cattgcatac gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat
     4261 gaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt acggggtcat
     4321 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     4381 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     4441 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     4501 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     4561 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     4621 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     4681 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     4741 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     4801 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt
     4861 tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac
     4921 accgggaccg atccagcctc cgcggccggg aacggtgcat tggaacgcgg attccccgtg
     4981 ccaagagtga cgtaagtacc gcctatagag tctataggcc cacccccttg gcttcgttag
     5041 aacgcggcta caattaatac ataaccttat gtatcataca catacgattt aggtgacact
     5101 atagaataac atccactttg cctttctctc cacaggtgtc cactcccagg tccaactgca
     5161 cctcggttct atcgaaaacg cgcctgctag cgccaccatg aagaccatca ttgccctgag
     5221 ctacatcttc tgcctggtgt tcgctgcggc cgccgttcca ggcgaagctg aacaacctgc
     5281 tcctgagctg gttgaggtgg aagtgggatc tacagccctg ctgaagtgtg gcctgagcca
     5341 gtctcagggc aatctgagcc acgtggactg gttcagcgtg cacaaagaga agcggaccct
     5401 gatcttcaga gtgcggcaag gacagggaca gagcgagcct ggcgaatacg agcaaagact
     5461 gagcctgcag gatagaggcg ccacactggc tctgacacaa gtgacccctc aggacgagcg
     5521 gatcttcctg tgccaaggca agaggcccag aagccaagag tacagaatcc agctgcgggt
     5581 gtacaaggcc cctgaggaac ccaacatcca agtgaaccct ctgggcatcc ccgtgaacag
     5641 caaagaacct gaagaggtgg ccacatgcgt gggcagaaac ggctacccca ttcctcaagt
     5701 gatctggtac aagaacggca ggcctctgaa agaggaaaag aaccgggtcc acatccagag
     5761 cagccagaca gtggaaagca gcggcctgta cacactgcag agcattctga aggcccagct
     5821 ggtcaaagag gacaaggacg cccagttcta ctgcgagctg aactacagac tgcccagcgg
     5881 caaccacatg aaggaatcca gagaagtgac cgtgcctgtg ttctacccca ccgagaaagt
     5941 gtggctggaa gtggaacccg tgggcatgct gaaagaaggc gacagagtcg agatcagatg
     6001 cctggccgat ggcaaccctc ctccacactt ttccatcagc aagcagaacc ccagcaccag
     6061 agaggccgag gaagaaacca ccaacgacaa tggcgtgctg gtgctcgagc ctgccagaaa
     6121 agaacacagc ggcagatacg agtgccaggg cctcgatctg gataccatga tcagcctgct
     6181 gagcgagcca caagagctgc tggtcaacta tgtgtccgat gtgcgagtgt ctcccgccgc
     6241 tcctgagaga caagagggaa gctctctgac actgacatgc gaggccgagt ccagccagga
     6301 cctggaattt cagtggctga gagaagagac aggccaggtg ctggaaagag gccctgttct
     6361 gcagctccac gatctgaaga gagaagccgg cggaggctac agatgtgtgg cctctgttcc
     6421 tagcatcccc ggcctgaata gaacccagct cgtgaacgtg gccatcttcg gacctccatg
     6481 gatggccttc aaagagcgga aagtctgggt caaagaaaac atggtgctga acctgagctg
     6541 cgaggcctct ggacacccta gacctaccat cagctggaat gtgaatggca ccgccagcga
     6601 gcaggatcag gaccctcaaa gagtgctgag caccctgaat gtgctggtca cccctgaact
     6661 gctggaaacc ggcgtggaat gtaccgccag caacgacctg ggcaagaaca ccagcatcct
     6721 gtttctggaa ctggtcaacc tgaccacact gacccctgac agcaatacca ccacaggcct
     6781 gagcacaagc accgcctctc cacacacaag agccaatagc acctccaccg agagaaagct
     6841 gcctgagcct gaaagccgcg gaggcgcgcc gggcggcgac aagacccaca cctgcccccc
     6901 ctgcccagct ccagaactgc tgggagggcc ttccgtgttc ctgttccccc ccaagcccaa
     6961 ggacaccctg atgatcagca ggacccccga ggtgacctgc gtggtggtgg ccgtgtccca
     7021 cgaggaccca gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa
     7081 gaccaagccc agagaggagc agtacaacag cacctacagg gtggtgtccg tgctgaccgt
     7141 gctgcaccag gactggctga acggcaaaga atacaagtgc aaagtctcca acaaggccct
     7201 ggctgcccca atcgaaaaga caatcagcaa ggccaagggc cagccacggg agccccaggt
     7261 gtacaccctg ccccccagcc gggaggagat gaccaagaac caggtgtccc tgacctgtct
     7321 ggtgaagggc ttctacccca gcgatatcgc cgtggagtgg gagagcaacg gccagcccga
     7381 gaacaactac aagaccaccc ccccagtgct ggacagcgac ggcagcttct tcctgtacag
     7441 caagctgacc gtggacaagt ccaggtggca gcagggcaac gtgttcagct gcagcgtgat
     7501 gcacgaggcc ctgcacaacc actacaccca gaagtccctg agcctgagcc ccggcggcag
     7561 cggtcttaat gatattttcg aggctcagaa gattgagtgg catgaacacc accaccatca
     7621 ccactaaggc cggcctgtag tcactggcac aacgcgttac ccggtaagcc aatcgggtat
     7681 acacggtcgt catactgcag acagggttct tcta
//
