LOCUS       62056_536        9294 bp DNA     circular SYN 18-OCT-2021
DEFINITION  Cloning vector CMV+ mScarlet-I with onboard mCitrine cassette, 
            complete sequence.
ACCESSION   MW987532
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9294)
  AUTHORS   Yang J, Lee J, Land MA, Lai S, Igoshin O, St-Pierre F.
  TITLE     A synthetic circuit for buffering gene dosage variation between 
            individual mammalian cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9294)
  AUTHORS   Yang J, Lee J, Land MA, Lai S, Igoshin O, St-Pierre F.
  TITLE     Direct Submission
  JOURNAL   Submitted (19-APR-2021) Neuroscience, Baylor College of Medicine, 
            One Baylor Plaza, Suite S636, Houston, Texas 77030, United States
REFERENCE   3  (bases 1 to 9294)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (19-APR-2021) Neuroscience, Baylor College of Medicine, One Baylor 
            Plaza, Suite S636, Houston, Texas 77030, United States"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
            On Oct 18, 2021 this sequence version replaced MW987532.1.
FEATURES             Location/Qualifiers
     source          1..9294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        163..360
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     protein_bind    361..379
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          588..1160
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             1178..1873
                     /codon_start=1
                     /label=mScarlet-I
                     /note="bright monomeric red fluorescent protein evolved
                     from a synthetic template (Bindels et al., 2016)"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     polyA_signal    2317..2541
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2825..2872
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2880..3899
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4032..4165
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4202..4218)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4226..4242)
                     /label=lac operator
                     /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(4250..4280)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4295..4316)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        4431..5612
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             5634..6350
                     /codon_start=1
                     /label=mEYFP
                     /note="enhanced YFP with monomerizing A206K mutation
                     (Zacharias et al., 2002)"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(6372..6390)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     rep_origin      complement(7086..7674)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7848..8705)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(8706..8810)
                     /label=AmpR promoter
     enhancer        9076..9294
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
       61 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      121 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      181 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      241 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      301 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      361 tccctatcag tgatagagat cagatctccc tatcagtgat agagagctgt tttgacctcc
      421 atagaagaca ccgggaccga tccagcctcc ggactctagc gtttaaactt aagctgggta
      481 cccggggatc ctctagggcc tctgagctat tccagaagta gtgaagaggc ttttttggag
      541 gcctaggctt ttgcaaaaag ctccggatcg atcctgagaa cttcagggtg agtttgggga
      601 cccttgattg ttctttcttt ttcgctattg taaaattcat gttatatgga gggggcaaag
      661 ttttcagggt gttgtttaga atgggaagat gtcccttgta tcaccatgga ccctcatgat
      721 aattttgttt ctttcacttt ctactctgtt gacaaccatt gtctcctctt attttctttt
      781 cattttctgt aactttttcg ttaaacttta gcttgcattt gtaacgaatt tttaaattca
      841 cttttgttta tttgtcagat tgtaagtact ttctctaatc actttttttt caaggcaatc
      901 agggtatatt atattgtact tcagcacagt tttagagaac aattgttata attaaatgat
      961 aaggtagaat atttctgcat ataaattctg gctggcgtgg aaatattctt attggtagaa
     1021 acaactacat cctggtcatc atcctgcctt tctctttatg gttacaatga tatacactgt
     1081 ttgagatgag gataaaatac tctgagtcca aaccgggccc ctctgctaac catgttcatg
     1141 ccttcttctt tttcctacag gtcctgcagg cgccaccatg gtgagcaagg gcgaggcagt
     1201 gatcaaggag ttcatgcggt tcaaggtgca catggagggc tccatgaacg gccacgagtt
     1261 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     1321 ggtgaccaag ggtggccccc tgcccttctc ctgggacatc ctgtcccctc agttcatgta
     1381 cggctccagg gccttcatca agcaccccgc cgacatcccc gactactata agcagtcctt
     1441 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt
     1501 gacccaggac acctccctgg aggacggcac cctgatctac aaggtgaagc tccgcggcac
     1561 caacttccct cctgacggcc ccgtaatgca gaagaagaca atgggctggg aagcgtccac
     1621 cgagcggttg taccccgagg acggcgtgct gaagggcgac attaagatgg ccctgcgcct
     1681 gaaggacggc ggccgctacc tggcggactt caagaccacc tacaaggcca agaagcccgt
     1741 gcagatgccc ggcgcctaca acgtcgaccg caagttggac atcacctccc acaacgagga
     1801 ctacaccgtg gtggaacagt acgaacgctc cgagggccgc cactccaccg gcggcatgga
     1861 cgagctgtac aagtaagcgg ccgcaatcaa cctctggatt acaaaatttg tgaaagattg
     1921 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     1981 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     2041 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     2101 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     2161 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     2221 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcctcgagt
     2281 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     2341 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2401 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2461 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     2521 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     2581 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2641 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2701 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2761 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2821 cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact tccttggcca
     2881 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     2941 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     3001 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     3061 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     3121 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     3181 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     3241 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     3301 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     3361 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     3421 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     3481 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     3541 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     3601 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     3661 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     3721 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     3781 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     3841 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     3901 agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3961 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4021 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4081 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4141 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4201 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4261 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4321 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4381 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct gctccggtgc
     4441 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     4501 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     4561 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     4621 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
     4681 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacgcc
     4741 cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt
     4801 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc
     4861 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata
     4921 agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata
     4981 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg
     5041 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc
     5101 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg
     5161 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt
     5221 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc
     5281 gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag
     5341 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct
     5401 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc
     5461 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct
     5521 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc
     5581 aaagtttttt tcttccattt caggtgtcgt gacataaact gccctagacc accatggtga
     5641 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     5701 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     5761 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     5821 ccaccttcgg ctacggcctg atgtgcttcg cccgctaccc cgaccacatg aagcagcacg
     5881 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     5941 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     6001 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     6061 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     6121 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     6181 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     6241 gctaccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     6301 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tagtaatcta
     6361 gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagt ccatagtcga
     6421 cggatccacc ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     6481 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     6541 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     6601 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     6661 caatgtatct taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     6721 tttgttaaat cagctcattt tttaaccaat aggccggctg tctgttcagt tatgaagatg
     6781 ccgcatagtt aagccagtat ctgctccctg gtcttctcca tcggatcctg atcaggggag
     6841 ctcacgggga cagccccccc ccaaagcccc cagggatgta attacgtccc tcccccgcta
     6901 gggggcagca gcgaccgccc ggggctccgc tccggtccgg cgctcccccc gcatcccgag
     6961 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatgc ggtgggctct
     7021 atggacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     7081 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     7141 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     7201 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     7261 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     7321 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     7381 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     7441 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     7501 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     7561 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     7621 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     7681 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     7741 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     7801 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     7861 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     7921 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     7981 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     8041 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     8101 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     8161 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     8221 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     8281 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     8341 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     8401 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     8461 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     8521 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     8581 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     8641 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     8701 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     8761 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     8821 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     8881 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     8941 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     9001 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     9061 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     9121 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     9181 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     9241 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaa
//
