LOCUS       sequence-440022-        7376 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible empty vector (transient mammalian expression) 
            with a C-terminal TwinStrep tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7376)
  TITLE     Harper Lab plasmids
REFERENCE   2  (bases 1 to 7376)
  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"
COMMENT     Sequence Label: pcDNA3.1-ccdB-TwinStrep
FEATURES             Location/Qualifiers
     source          1..7376
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    969..1093
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1118..1123,1124..1141,1142..1148)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1202..1861
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1268..1287)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2203..2508
                     /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     2387..2406
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2549..2673)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(2675..2680,2681..2704,2705..2740,2741..2764)
                     /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"
     primer_bind     complement(2808..2825)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2814..2925
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3074..3502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3161..3180)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3371..3392
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3511..3531)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3516..3845
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3696..3831
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3758..3777
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3894..4919
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3894..4919
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    5049..5170
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5086..5105)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5140..5159
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5219..5235)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5219..5235)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5232..5254)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5243..5259
                     /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(5267..5273,5274..5291,5292..5297))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5312..5333
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5450..5467)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5621..6209)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5701..5720)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6380..7171,7172..7240))
                     /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     7003..7022
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7241..7345)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc
      961 agatatcaac aagtttgtac aaaaaagctg aacgagaaac gtaaaatgat ataaatatca
     1021 atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa acacaacata
     1081 tccagtcact atggcggccg cattaggcac cccaggcttt acactttatg cttccggctc
     1141 gtataatgtg tggattttga gttaggatcc gtcgagattt tcaggagcta aggaagctaa
     1201 aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga
     1261 acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga
     1321 tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat
     1381 tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg
     1441 tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga
     1501 aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata
     1561 ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga
     1621 gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt
     1681 ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg
     1741 cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca
     1801 tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta
     1861 aagatctgga tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt
     1921 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct
     1981 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
     2041 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
     2101 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
     2161 aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac
     2221 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     2281 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     2341 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     2401 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
     2461 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc
     2521 cttatacaca gccagtctgc aggtcgacca tagtgactgg atatgttgtg ttttacagta
     2581 ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta
     2641 cgtttctcgt tcagctttct tgtacaaagt ggttagtgca tggagccatc cgcagtttga
     2701 aaaaggtggt ggtagcggtg gtggttcagg tggtagtgca tggtcacacc ctcagtttga
     2761 gaaatgataa ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     2821 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2881 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2941 gtgtcattct attctggggg gtggggtggg gcaggacagc aaggggaaga caatagcagg
     3001 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3061 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3121 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3181 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3241 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3301 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3361 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3421 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3481 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3541 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3601 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3661 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3721 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3781 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3841 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgatgaaaa
     3901 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     3961 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     4021 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     4081 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     4141 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     4201 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     4261 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     4321 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     4381 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     4441 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     4501 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     4561 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     4621 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     4681 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     4741 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     4801 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     4861 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     4921 acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4981 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5041 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5101 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5161 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5221 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5281 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5341 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5401 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5461 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5521 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5581 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5641 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5701 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5761 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5821 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5881 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5941 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6001 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6061 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6121 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6181 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6241 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6301 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6361 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6421 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6481 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6541 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6601 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6661 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6721 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6781 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6841 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6901 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6961 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7021 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7081 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7141 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7201 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7261 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7321 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
