LOCUS       sequence-433197-        7915 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encoding a fluorescent reporter for ERK activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7915)
  TITLE     Collective cell migration is coordinated by front-biased activation 
            of the feed-forward loop consisting of Ras, Rab5, and Rac1.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7915)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pPBpuro-ERK-KTR-mCherry
FEATURES             Location/Qualifiers
     source          1..7915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        1011..1390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1392..1669
                     /label=chicken beta-actin promoter
     intron          1670..2686
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2610..2632
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2694..2713
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(3159..3177)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3427..3446
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3821..4370
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3965..3982)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4192..4211
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4373..4972
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4373..4392)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4869..4889
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4999..5016)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5005..5229
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5276..5292)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5300..5316
                     /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(5324..5330,5331..5348,5349..5354))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5369..5390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   5624..5686
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5727..5747)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5727..5745)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5766..5782)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5790..5806
                     /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(5814..5820,5821..5838,5839..5844))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5859..5880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5997..6014)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6168..6756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6248..6267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6927..7718,7719..7787))
                     /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     7550..7569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7788..7892)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcatg ggcccatctc gacattgatt
     1021 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     1081 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1141 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     1201 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     1261 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     1321 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     1381 tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
     1441 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
     1501 gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag
     1561 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
     1621 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
     1681 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     1741 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
     1801 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
     1861 gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg
     1921 cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg
     1981 cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc
     2041 gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
     2101 gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg
     2161 agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct
     2221 cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg
     2281 ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg
     2341 cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc
     2401 tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg
     2461 gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc
     2521 gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg
     2581 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc
     2641 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt
     2701 gctggttatt gtgctgtctc atcattttgg caaagaattg ggatccgaat tcgccaccat
     2761 gaaaggccgc aagcctcgcg atctggagtt gcctctcagc cctagcctcc tggggggaca
     2821 gggccctgag cgcacacccg ggtccggcac ctctagtggt ttgcaggctc cgggacctgc
     2881 cctgtcaccc tccaagcgca gcggcctgga ggatcctgcc acacccagca aaaaaccaag
     2941 gaccccctca gtcagctcgc ggttagagcg actgacgctc cagtcttctt tccaattccc
     3001 gtccggcggc cgcatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     3061 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     3121 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     3181 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     3241 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtcctccc ccgagggctt
     3301 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     3361 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     3421 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     3481 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     3541 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     3601 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     3661 ggaacagtac gaacgcgccg agggccgcca ctccactctc ggcatggacg agctgtctag
     3721 aggatccgtt aactaactcg agggcggccg ctctagagtc aactaactta agctagcaac
     3781 ggtttccctc tagcgggatc aattccgccc ccccccccta acgttactgg ccgaagccgc
     3841 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     3901 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     3961 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
     4021 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     4081 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     4141 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     4201 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     4261 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
     4321 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgataata ccatgaccga
     4381 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     4441 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     4501 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     4561 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     4621 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     4681 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     4741 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     4801 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     4861 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     4921 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gatcgatgat
     4981 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     5041 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     5101 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     5161 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     5221 gctctatggc ttctgaggcg gaaagaacct gcagcccaag cttggcgtaa tcatggtcat
     5281 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5341 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5401 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatctta aaagttttgt
     5461 tactttatag aagaaatttt gagtttttgt ttttttttaa taaataaata aacataaata
     5521 aattgtttgt tgaatttatt attagtatgt aagtgtaaat ataataaaac ttaatatcta
     5581 ttcaaattaa taaataaacc tcgatataca gaccgataaa acacatgcgt caattttacg
     5641 catgattatc tttaacgtac gtcacaatat gattatcttt ctagggttaa tctagtatac
     5701 gcgtttaatt aatccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     5761 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5821 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5881 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5941 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6001 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6061 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6121 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6181 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6241 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6301 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6361 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6421 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6481 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6541 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6601 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6661 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6721 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6781 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6841 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6901 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6961 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7021 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7081 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7141 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7201 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7261 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7321 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7381 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7441 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7501 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7561 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7621 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7681 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7741 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7801 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7861 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac
//
