LOCUS       Exported                5555 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pCS2 CMV expression vector driving V5-tagged mScarlet-I-fused human 
            KRAS4A.
ACCESSION   .
VERSION     .
KEYWORDS    JDW 836 (pCS2-V5-mScarlet-I-hsKRAS4A-WT)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5555)
  AUTHORS   Fish JE, Flores-Suarez CP, Boudreau E, Herman AM, Gutierrez MC, 
            Gustafson D, DiStefano PV, Cui M, Chen Z, Berman De Ruiz K, 
            Schexnayder TS, Ward CS, Radovanovic I, Wythe JD
  TITLE     Somatic Gain of KRAS Function in the Endothelium is Sufficient to 
            Cause Vascular Malformations that Require MEK but not PI3K 
            Signaling.
  JOURNAL   Circ Res. 2020 Jun 17. doi: 10.1161/CIRCRESAHA.119.316500.
  PUBMED    32552404
REFERENCE   2  (bases 1 to 5555)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5555
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        859..877
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     859..876
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    932..956
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      975..984
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             984..1025
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1041..1736
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1174..1192)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1442..1461
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2362..2386)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2409..2426)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2431..2565
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2455..2474)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2509..2528
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2678..2698)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2678..2696)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2717..2733)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2717..2733)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2730..2752)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2741..2757
                     /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(2765..2795)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2810..2831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2948..2965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3119..3707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3199..3218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3878..4738)
                     /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     4501..4520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4739..4843)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4869..5324)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5006..5027)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5218..5237
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5402..824
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 attcaatatg gcggacctgg cactgtgcca actggggagg ggtctacttg gcacggtgcc
       61 aagtttgagg aggggtcttg gccctgtgcc aagtccgcca tattgaattg gcatggtgcc
      121 aataatggcg gccatattgg ctatatgcca ggatcaatat ataggcaata tccaatatgg
      181 ccctatgcca atatggctat tggccaggtt caatactatg tattggccct atgccatata
      241 gtattccata tatgggtttt cctattgacg tagatagccc ctcccaatgg gcggtcccat
      301 ataccatata tggggcttcc taataccgcc catagccact cccccattga cgtcaatggt
      361 ctctatatat ggtctttcct attgacgtca tatgggcggt cctattgacg tatatggcgc
      421 ctcccccatt gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc attgacgtca
      481 ataggaccac ccaccattga cgtcaatggg atggctcatt gcccattcat atccgttctc
      541 acgcccccta ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat caatatctat
      601 taatagtaac ttggcaagta cattactatt ggaagtacgc cagggtacat tggcagtact
      661 cccattgacg tcaatggcgg taaatggccc gcgatggctg ccaagtacat ccccattgac
      721 gtcaatgggg aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg ggcggtaggc
      781 gtgcctaatg ggaggtctat ataagcaatg ctcgtttagg gaaccgccat tctgcctggg
      841 gacgtcggag caagcttgat ttaggtgaca ctatagaata caagctactt gttctttttg
      901 caggatccca tcgattcgaa ttcaaggatc aacaagtttg tacaaaaaag caggctggat
      961 ccactagtgc tagcgccacc atgggtaagc ctatccctaa ccctctcctc ggtctcgatt
     1021 ctacgggtgg tgctggtggt atggtgagca agggcgaggc agtgatcaag gagttcatgc
     1081 ggttcaaggt gcacatggag ggctccatga acggccacga gttcgagatc gagggcgagg
     1141 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     1201 ccctgccctt ctcctgggac atcctgtccc ctcagttcat gtacggctcc agggccttca
     1261 tcaagcaccc cgccgacatc cccgactact ataagcagtc cttccccgag ggcttcaagt
     1321 gggagcgcgt gatgaacttc gaggacggcg gcgccgtgac cgtgacccag gacacctccc
     1381 tggaggacgg caccctgatc tacaaggtga agctccgcgg caccaacttc cctcctgacg
     1441 gccccgtaat gcagaagaag acaatgggct gggaagcatc caccgagcgg ttgtaccccg
     1501 aggacggcgt gctgaagggc gacattaaga tggccctgcg cctgaaggac ggcggccgct
     1561 acctggcgga cttcaagacc acctacaagg ccaagaagcc cgtgcagatg cccggcgcct
     1621 acaacgtcga ccgcaagttg gacatcacct cccacaacga ggactacacc gtggtggaac
     1681 agtacgaacg ctccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtact
     1741 cctccggact cagatctgga ggctccggag gcatgactga atataaactt gtggtagttg
     1801 gagctggtgg cgtaggcaag agtgccttga cgatacagct aattcagaat cattttgtgg
     1861 acgaatatga tccaacaata gaggattcct acaggaagca agtagtaatt gatggagaaa
     1921 cctgtctctt ggatattctc gacacagcag gtcaagagga gtacagtgca atgagggacc
     1981 agtacatgag gactggggag ggctttcttt gtgtatttgc cataaataat actaaatcat
     2041 ttgaagatat tcaccattat agagaacaaa ttaaaagagt taaggactct gaagatgtac
     2101 ctatggtcct agtaggaaat aaatgtgatt tgccttctag aacagtagac acaaaacagg
     2161 ctcaggactt agcaagaagt tatggaattc cttttattga aacatcagca aagacaagac
     2221 agagagtgga ggatgctttt tatacattgg tgagggagat ccgacaatac agattgaaaa
     2281 aaatcagcaa agaagaaaag actcctggct gtgtgaaaat taaaaaatgc attataatgt
     2341 aaggcgcgcc ctcgagagat ctcccagctt tcttgtacaa agtggttgat cctctcgagc
     2401 ctctagaact atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag
     2461 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     2521 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     2581 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cgcggccgcg
     2641 gcgccaatgc attgggcccg gtacccagct tttgttccct ttagtgaggg ttaattgcgc
     2701 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2761 cacacaacat acgagccggg agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2821 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2881 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2941 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3001 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3061 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3121 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3181 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3241 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3301 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3361 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3421 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3481 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3541 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3601 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3661 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3721 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3781 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3841 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3901 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3961 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4021 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4081 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4141 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4201 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4261 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4321 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4381 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4441 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4501 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4561 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4621 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4681 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4741 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4801 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4861 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     4921 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     4981 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     5041 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     5101 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     5161 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     5221 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     5281 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag
     5341 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagtcgac
     5401 cgccaattca atatggcgta tatggactca tgccaattca atatggtgga tctggacctg
     5461 tgccaattca atatggcgta tatggactcg tgccaattca atatggtgga tctggacccc
     5521 agccaattca atatggcgga cttggcacca tgcca
//
