LOCUS       sequence-67169-e        6964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This is a Gateway destination vector for secreted expression in 
            Drosophila cell culture by CuSO4 induction. Inserts will be 
            pentamerized and tagged with Alkaline phosphatase, FLAG and His 
            tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6964)
  AUTHORS   Ozkan E, Carrillo RA, Eastman CL, Weiszmann R, Waghray D, Johnson 
            KG, Zinn K, Celniker SE, Garcia KC
  TITLE     An Extracellular Interactome of Immunoglobulin and LRR Proteins 
            Reveals Receptor-Ligand Networks.
  JOURNAL   Cell. 2013 Jul 3;154(1):228-39. doi: 10.1016/j.cell.2013.06.006.
  PUBMED    23827685
REFERENCE   2  (bases 1 to 6964)
  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.2013.06"; journalName: "Cell"; date: "2013-07-3- 3";
            volume: "154"; issue: "1"; pages: "228-39"
COMMENT     Sequence Label: pECIA14
FEATURES             Location/Qualifiers
     source          1..6964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        401..827
                     /label=MT promoter
                     /note="Drosophila metallothionein promoter"
     sig_peptide     845..898
                     /codon_start=1
                     /product="Drosophila BiP signal sequence"
                     /label=BiP signal sequence
                     /translation="MKLCILLAVVAFVGLSLG"
     protein_bind    908..1032
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1057..1062,1063..1080,1081..1087)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1141..1800
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1207..1226)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2142..2447
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2326..2345
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2488..2612)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2638..2661
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4336..4359
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4375..4392
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4415..4432)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4465..4599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4489..4508)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4543..4562
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4743..4759)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4743..4759)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4756..4778)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4767..4783
                     /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)."
     promoter        complement(join(4791..4797,4798..4815,4816..4821))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4836..4857
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4974..4991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5145..5733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5225..5244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5904..6695,6696..6764))
                     /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     6527..6546
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6765..6869)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6937..6955
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt aattcgttgc aggacaggat
      421 gtggtgcccg atgtgactag ctctttgctg caggccgtcc tatcctctgg ttccgataag
      481 agacccagaa ctccggcccc ccaccgccca ccgccacccc catacatatg tggtacgcaa
      541 gtaagagtgc ctgcgcatgc cccatgtgcc ccaccaagag ctttgcatcc catacaagtc
      601 cccaaagtgg agaaccgaac caattcttcg cgggcagaac aaaagcttct gcacacgtct
      661 ccactcgaat ttggagccgg ccggcgtgtg caaaagaggt gaatcgaacg aaagacccgt
      721 gtgtaaagcc gcgtttccaa aatgtataaa accgagagca tctggccaat gtgcatcagt
      781 tgtggtcagc agcaaaatca agtgaatcat ctcagtgcaa ctaaaggggg gatccgatct
      841 caatatgaag ttatgcatat tactggccgt cgtggccttt gttggcctct cgctcgggag
      901 atcatcaaca agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa
      961 tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat
     1021 ccagtcacta tggcggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg
     1081 tataatgtgt ggattttgag ttaggatccg tcgagatttt caggagctaa ggaagctaaa
     1141 atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa
     1201 cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat
     1261 attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt
     1321 cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt
     1381 gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa
     1441 acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat
     1501 tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag
     1561 aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg
     1621 gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc
     1681 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat
     1741 gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa
     1801 agatctggat ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt
     1861 tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta
     1921 tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata
     1981 tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg
     2041 tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga
     2101 aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca
     2161 cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca
     2221 cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct
     2281 cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg
     2341 atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg
     2401 aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc
     2461 ttatacacag ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat
     2521 tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac
     2581 gtttctcgtt cagctttctt gtacaaagtg gttgatatcc agcacagtgg cggccgcctg
     2641 gaggtgctgt tccagggacc aagcggcaat cccggcggcg atctggcccc ccagatgctg
     2701 cgcgagctgc aggagaccaa tgccgccctg caggacgtgc gcgagctgct gcgccaccgc
     2761 gtgaaggaga tcaccttcct gaagaacacc gtgatggagt gcgacgcctg cggcatgcag
     2821 ccctctagag gctccggctc catcatccca gttgaggagg agaacccgga cttctggaac
     2881 cgcgaggcag ccgaggccct gggtgccgcc aagaagctgc agcctgcaca gacagccgcc
     2941 aagaacctca tcatcttcct gggcgatggg atgggggtgt ctacggtgac agctgccagg
     3001 atcctaaaag ggcagaagaa ggacaaactg gggcctgaga tacccctggc catggaccgc
     3061 ttcccatatg tggctctgtc caagacatac aatgtagaca aacatgtgcc agacagtgga
     3121 gccacagcca cggcctacct gtgcggggtc aagggcaact tccagaccat tggcttgagt
     3181 gcagccgccc gctttaacca gtgcaacacg acacgcggca acgaggtcat ctccgtgatg
     3241 aatcgggcca agaaagcagg gaagtcagtg ggagtggtaa ccaccacacg agtgcagcac
     3301 gcctcgccag ccggcaccta cgcccacacg gtgaaccgca actggtactc ggacgccgac
     3361 gtgcctgcct cggcccgcca ggaggggtgc caggacatcg ctacgcagct catctccaac
     3421 atggacattg acgtgatcct aggtggaggc cgaaagtaca tgtttcgcat gggaacccca
     3481 gaccctgagt acccagatga ctacagccaa ggtgggacca ggctggacgg gaagaatctg
     3541 gtgcaggaat ggctggcgaa gcgccagggt gcccggtatg tgtggaaccg cactgagctc
     3601 atgcaggctt ccctggaccc gtctgtgacc catctcatgg gtctctttga gcctggagac
     3661 atgaaatacg agatccaccg agactccaca ctggacccct ccctgatgga gatgacagag
     3721 gctgccctgc gcctgctgag caggaacccc cgcggcttct tcctcttcgt ggagggtggt
     3781 cgcatcgacc atggtcatca tgaaagcagg gcttaccggg cactgactga gacgatcatg
     3841 ttcgacgacg ccattgagag ggcgggccag ctcaccagcg aggaggacac gctgagcctc
     3901 gtcactgccg accactccca cgtcttctcc ttcggaggct accccctgcg agggagctcc
     3961 atcttcgggc tggcccctgg caaggcccgg gacaggaagg cctacacggt cctcctatac
     4021 ggaaacggtc caggctatgt gctcaaggac ggcgcccggc cggatgttac cgagagcgag
     4081 agcgggagcc ccgagtatcg gcagcagtca gcagtgcccc tggacgaaga gacccacgca
     4141 ggcgaggacg tggcggtgtt cgcgcgcggc ccgcaggcgc acctggttca cggcgtgcag
     4201 gagcagacct tcatagcgca cgtcatggcc ttcgccgcct gcctggagcc ctacaccgcc
     4261 tgcgacctgg cgccccccgc cggcaccacc gacgccgcgc acccgggttc tagaggaagc
     4321 acctccggcg ataacgatta caaggatgat gatgataagg ccagcggcac cggtcatcat
     4381 caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc taaggcctga
     4441 gctcgctgat cagcctcgat cgaggatcca gacatgataa gatacattga tgagtttgga
     4501 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4561 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4621 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     4681 aaatgtggta tggctgatta tgatcagtcg acctgcaggc atgcaagctt ggcgtaatca
     4741 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4801 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4861 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4921 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4981 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5041 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5101 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5161 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5221 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5281 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5341 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5401 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5461 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5521 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5581 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5641 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5701 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5761 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5821 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5881 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5941 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6001 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6061 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6121 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6181 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6241 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6301 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6361 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6421 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6481 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6541 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6601 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6661 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6721 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6781 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6841 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6901 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     6961 cgtc
//
