LOCUS       sequence-85571-e        5307 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for parallel SLIC cloning containing 
            N-terminal HA, 8x His and FLAG tags. C-terminal tags can be added 
            during SLIC..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5307)
  AUTHORS   Scholz J, Besir H, Strasser C, Suppmann S
  TITLE     A new method to customize protein expression vectors for fast, 
            efficient and background free parallel cloning.
  JOURNAL   BMC Biotechnol. 2013 Feb 14;13(1):12.
  PUBMED    23410102
REFERENCE   2  (bases 1 to 5307)
  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"; journalName: "BMC 
            Biotechnol."; date: "2013-02-14"; volume: "13(1)"; pages: "12"
COMMENT     Sequence Label: pCoofy50
FEATURES             Location/Qualifiers
     source          1..5307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        106..1287
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          337..1278
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1235..1255
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1359..1385
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1398..1421
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1431..1454
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1461..1484
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             1521..1529
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1531..1839
                     /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="MIQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSD
                     KVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1718..1737
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1843..1872
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     polyA_signal    1977..2371
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     primer_bind     complement(2682..2699)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2682..2698)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2691..2713)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2907..2926)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3026..3048
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3086..3104)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3172..3276
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3277..3345,3346..4137)
                     /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     complement(3495..3514)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4308..4896
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4797..4816
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5050..5067
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5184..5205
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5220..5225,5226..5243,5244..5250)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5258..5274
                     /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)."
     primer_bind     5263..5285
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5282..5298
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5282..5298
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gaattaattc gagctcgccc aactttgcaa agatggataa agttttaaac agagaggaat
       61 ctttgcagct aatggacctt ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt
      121 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
      181 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
      241 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
      301 gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc
      361 gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc cacgcccctg
      421 gctgcaatac gtgaatcttg atcccgagct tcgggttgga agtgggtggg agagttcgag
      481 gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
      541 gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc
      601 tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct
      661 tgtaaatgcg ggccaagatc tgcacactgt atttcggttt ttggggccgc gggcggcgac
      721 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
      781 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccc
      841 ccgtgtatcg ccccgccctg ggcggcaagg ttggcccggt cggcaccagt tgcgtgagcg
      901 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
      961 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     1021 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctggatta gttctcgagc
     1081 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     1141 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     1201 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     1261 ttttttcttc catttcaggt gtcgtgagga attagcttgc ttacatttgc ttctgacaca
     1321 actgtgttca ctagcaacct caaacagaca ccatgaagta cccttacgat gtgcccgatt
     1381 acgcccccat gggatcccat caccatcacc atcaccatca cggtggttcc gattacaagg
     1441 atgacgatga caaggaattc ctggaagttc tgttccaggg gcccttgaca acataaaaac
     1501 tttgtgttat acttgtaacg taaggaggta atgattcagt ttaaggttta cacctataaa
     1561 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     1621 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     1681 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     1741 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     1801 atcaaaaacg ccattaacct gatgttctgg ggaatataag agcatcatca tcatcaccac
     1861 caccaccacc actgagcggc cgctttcgaa tctagaaatt caccccacca gtgcaggctg
     1921 cctatcagaa agtggtggct ggtgtggcta atgccctggc ccacaagtat cactaagctc
     1981 gctttcttgc tgtccaattt ctattaaagg ttcctttgtt ccctaagtcc aactactaaa
     2041 ctgggggata ttatgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt
     2101 ttcattgcaa tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga
     2161 ggtcagtgca tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac
     2221 tatatcttaa actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca
     2281 gcccctgatg cctatgcctt attcatccct cagaaaagga ttcaagtaga ggcttgattt
     2341 ggaggttaaa gttttgctat gctgtatttt acattactta ttgttttagc tgtcctcatg
     2401 aatgtctttt cactacccat ttgcttatcc tgcatctctc agccttgact ccactcagtt
     2461 ctcttgctta gagataccac ctttcccctg aagtgttcct tccatgtttt acggcgagat
     2521 ggtttctcct cgcctggcca ctcagcctta gttgtctctg ttgtcttata gaggtctact
     2581 tgaagaagga aaaacagggg gcatggtttg actgtcctgt gagcccttct tccctgcctc
     2641 ccccactcac agtgacccgg aatctgcagc ccaagcttgg cactggccgt cgttttacaa
     2701 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     2761 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     2821 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     2881 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     2941 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3001 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3061 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     3121 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3181 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3241 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3301 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3361 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3421 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3481 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3541 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3601 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3661 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3721 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3781 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3841 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3901 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3961 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4021 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4081 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4141 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4201 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4261 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4321 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4381 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4441 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4501 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4561 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4621 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4681 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4741 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4801 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4861 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4921 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4981 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5041 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     5101 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     5161 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     5221 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     5281 acaggaaaca gctatgacca tgattac
//
