LOCUS       sequence-243401-        5294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian vector for parallel SLIC cloning containing N-terminal 
            TwinStrep and PreScission Cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5294)
  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 5294)
  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: pCoofy56
FEATURES             Location/Qualifiers
     source          1..5294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     342..359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    476..497
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(512..517,518..535,536..542)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    550..566
                     /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     555..577
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     574..590
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     574..590
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        704..1882
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          935..1873
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1830..1850
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(1951..1956,1957..1980,1981..2016,2017..2040)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             2047..2070
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             2107..2115
                     /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             2117..2425
                     /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     2304..2323
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2429..2458
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     polyA_signal    2563..2957
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     primer_bind     complement(3268..3285)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3268..3284)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3277..3299)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3493..3512)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3612..3634
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3672..3690)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3758..3862
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3863..3931,3932..4723)
                     /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(4081..4100)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4894..5294,1..188)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
       61 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      121 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      181 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      241 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
      301 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
      361 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
      421 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
      481 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
      541 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga
      601 attaattcga gctcgcccag ctttgcaaag atggataaag ttttaaacag agaggaatct
      661 ttgcagctaa tggaccttct aggtcttgaa aggagtggga attggctccg gtgcccgtca
      721 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
      781 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
      841 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
      901 tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg
      961 gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca
     1021 gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg
     1081 cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg
     1141 ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc
     1201 catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa
     1261 tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc
     1321 ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat
     1381 cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg
     1441 tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag
     1501 atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga
     1561 gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc
     1621 atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg
     1681 gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga
     1741 gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc
     1801 cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt
     1861 tcttccattt caggtgtcgt gaggaattag cttgcttaca tttgcttctg acacaactgt
     1921 gttcactagc aacctcaaac agacaccatg agcgcttgga gccacccgca gttcgaaaaa
     1981 ggtggaggtt ctggcggtgg atcgggaggt tcagcgtgga gccacccgca gttcgagaaa
     2041 gaattcctgg aagttctgtt ccaggggccc ttgacaacat aaaaactttg tgttatactt
     2101 gtaacgtaag gaggtaatga ttcagtttaa ggtttacacc tataaaagag agagccgtta
     2161 tcgtctgttt gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat
     2221 ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt
     2281 gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc
     2341 cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat
     2401 taacctgatg ttctggggaa tataagagca tcatcatcat caccaccacc accaccactg
     2461 agcggccgct ttcgaatcta gaaattcacc ccaccagtgc aggctgccta tcagaaagtg
     2521 gtggctggtg tggctaatgc cctggcccac aagtatcact aagctcgctt tcttgctgtc
     2581 caatttctat taaaggttcc tttgttccct aagtccaact actaaactgg gggatattat
     2641 gaagggcctt gagcatctgg attctgccta ataaaaaaca tttattttca ttgcaatgat
     2701 gtatttaaat tatttctgaa tattttacta aaaagggaat gtgggaggtc agtgcattta
     2761 aaacataaag aaatgaagag ctagttcaaa ccttgggaaa atacactata tcttaaactc
     2821 catgaaagaa ggtgaggctg caaacagcta atgcacattg gcaacagccc ctgatgccta
     2881 tgccttattc atccctcaga aaaggattca agtagaggct tgatttggag gttaaagttt
     2941 tgctatgctg tattttacat tacttattgt tttagctgtc ctcatgaatg tcttttcact
     3001 acccatttgc ttatcctgca tctctcagcc ttgactccac tcagttctct tgcttagaga
     3061 taccaccttt cccctgaagt gttccttcca tgttttacgg cgagatggtt tctcctcgcc
     3121 tggccactca gccttagttg tctctgttgt cttatagagg tctacttgaa gaaggaaaaa
     3181 cagggggcat ggtttgactg tcctgtgagc ccttcttccc tgcctccccc actcacagtg
     3241 acccggaatc tgcagcccaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga
     3301 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     3361 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     3421 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     3481 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     3541 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3601 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3661 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     3721 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3781 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3841 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3901 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3961 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4021 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4081 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4141 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4201 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4261 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4321 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4381 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     4441 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     4501 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4561 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4621 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4681 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4741 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4801 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4861 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4921 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttttttgc cggatcaaga
     4981 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5041 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5101 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5161 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5221 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5281 tgagctatga gaaa
//
