LOCUS       Exported                5990 bp ds-DNA     circular SYN 05-OCT-2020
DEFINITION  R4-R3 mammalian expression vector, puromycin resistance, downstream 
            polyA, no promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5990)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Oct 7, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
COMMENT     pDest-311 and pDest-305 were both digested in 2ug reactions with 
            Afl3 in Buffer 3 with BSA and 2uL of enzyme for 2 hours at 
            37degrees.
            Both digests were verified on a gel, and the pDest-311 digest was 
            cleaned.  It was then phosphatase treated for 15 minutes at 
            37degrees with 2ul of alkaline phosphatase.  It was then cleaned 
            again.
            The pDest-305 digest was gel extracted, with the 1.6kB piece being 
            cut out.  The entire digest was gel extracted, and then MinEluted in
            20uL of TE.  7/25/2011  JM
            Both pieces were verified on a gel to confirm concentrations.  A 
            ligation was done using 1uL of the phophatase treated backbone and 
            4uL of the insert piece.  This ligation was Xfed into DB3.1 cells 
            and was plated on A/C at 37degrees. 7/26/2011  JM
            There were over 200 colonies on the ligation plate, which indicates 
            that the phosphatase treatment might have failed.  However, 10 
            colonies were picked to see. 7/27/2011  JM
            All of the colonies were religated pDest-311, although clone 6 has a
            correct band in it also, and so this was kept just in case.
            The pDest-311 digest was phosphatase treated again, with 2ul of 
            phosphatase for 30minutes at 37degrees, and then was cleaned and gel
            confirmed again.
            A ligation was done using 1ul of this newly phosphatased backbone 
            vector and 4ul of the pDest-305 gel extracted insert.  This ligation
            was Xfed into DB3.1 cells and plated on A/C at 37degrees. 7/28/2011 
            JM
            There were about 75-100 colonies on the plate, which is less than 
            previously, but still more than expected. 7/29/2011  JM
            8 colonies were picked. Clones 1-3 and 5-8 were the correct size and
            were digested with EcoO109I to check for directionality of the 
            insert.  Clones 3, 5, 6, and 7 were in the forward orientation, and 
            were sent to sequencing. 8/2/2011  JM
            Clone 312.5 determined to be OK by sequencing with RiL/RiR (plate 
            PEL110804, wells G03/G04), DE 8/8/11
FEATURES             Location/Qualifiers
     source          1..5990
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    407..530
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(571..876)
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             complement(1218..1877)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(1931..1961)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(1986..2109)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2156..2172)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2180..2196
                     /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(2204..2234)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2249..2270
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    2511..2735
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2770..3241
                     /label=mPGK
     CDS             3255..3854
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    3910..4097
                     /label=C2pA
     rep_origin      complement(4171..4759)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4930..5790)
                     /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"
     promoter        complement(5791..5895)
                     /gene="bla"
                     /label=AmpR promoter
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 cgagatcaac tttgtataga
      421 aaagttgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
      481 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gtcgacctgc
      541 agactggctg tgtataaggg agcctgacat ttatattccc cagaacatca ggttaatggc
      601 gtttttgatg tcattttcgc ggtggctgag atcagccact tcttccccga taacggagac
      661 cggcacactg gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac
      721 cgggtaaagt tcacggggga ctttatctga cagcagacgt gcactggcca gggggatcac
      781 catccgtcgc ccgggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg
      841 gctctctctt ttataggtgt aaaccttaaa ctgcatttca ccagcccctg ttctcgtcgg
      901 caaaagagcc gttcatttca ataaaccggg cgacctcagc catcccttcc tgattttccg
      961 ctttccagcg ttcggcacgc agacgacggg cttcattctg catggttgtg cttaccgaac
     1021 cggagatatt gacatcatat atgccttgag caactgatag ctgtcgctgt caactgtcac
     1081 tgtaatacgc tgcttcatag catacctctt tttgacatac ttcgggtata catatcagta
     1141 tatattctta taccgcaaaa atcagcgcgc aaatacgcat actgttatct ggcttttagt
     1201 aagccggatc ctctagatta cgccccgccc tgccactcat cgcagtactg ttgtaattca
     1261 ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc
     1321 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag
     1381 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct
     1441 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa
     1501 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
     1561 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta
     1621 tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg agcattcatc
     1681 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc
     1741 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac
     1801 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
     1861 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgacgg atcctaactc
     1921 aaaatccaca cattatacga gccggaagca taaagtgtaa agcctggggt gcctaatgcg
     1981 gccgccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca
     2041 aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca actttattat
     2101 acatagttga tcctctagag tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat
     2161 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     2221 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     2281 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2341 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2401 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2461 ggttatccac agaatcaggg gataacgcag gaaagaacat gtagcctcga ctgtgccttc
     2521 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2581 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2641 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2701 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     2761 gggaagcttc taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt
     2821 agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac
     2881 atccaccggc cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt
     2941 ctactcctcc cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt
     3001 gacaaatgga agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat
     3061 ggaagcgggt aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg
     3121 ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc
     3181 tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt
     3241 tcgaccagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     3301 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     3361 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     3421 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     3481 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     3541 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     3601 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg
     3661 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     3721 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     3781 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     3841 agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac
     3901 cccatgcatc gtagaattcc ggacttgact catgcttgtt tcactttcac atggaatttc
     3961 ccagttatga aattaataaa aatcaatggt ttccacatct ctcagtgcct ctatctggag
     4021 gccaggtagg gctggccttg ggggaggggg aggccagaat gactccaaga gctacaggaa
     4081 ggcaggtcag agaccccaag cttgaattca catgtgagca aaaggccagc aaaaggccag
     4141 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4201 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4261 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4321 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4381 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4441 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4501 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4561 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     4621 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4681 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4741 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4801 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4861 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4921 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4981 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5041 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5101 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5161 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5221 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5281 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5341 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5401 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5461 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5521 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5581 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5641 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5701 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5761 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5821 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5881 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     5941 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
