LOCUS       sequence-450066-        8112 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Piggybac expression vector with inactive phospho-mutant (YYFF) of 
            Illusia FRET reporter for integrin phosphorylation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8112)
  TITLE     Dynamic regulation of integrin beta-1 phosphorylation supports 
            invasive breast cancer progression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8112)
  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            "Unpublished"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: pPB.DEST-Illusia(YYFF)
FEATURES             Location/Qualifiers
     source          1..8112
                     /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"
     intron          1668..2684
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2608..2630
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2692..2711
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2805..2814
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2871..2873,2874..2876,2877..3584)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLLCTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITEGMNELY"
     CDS             join(4368..4370,4371..4373,4374..5087)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     protein_bind    complement(5097..5121)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(5154..5171)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5160..5384
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5431..5447)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5455..5471
                     /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(5479..5485,5486..5503,5504..5509))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5524..5545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   5811..5873
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5924..5944)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5924..5942)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5963..5979)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5987..6003
                     /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(6011..6017,6018..6035,6036..6041))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6056..6077
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6194..6211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6365..6953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6445..6464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7124..7915,7916..7984))
                     /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     7747..7766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7985..8089)
                     /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 ggcgccgccc cgtgggggcg gggggggggc gggggggggg ggggggaggc
     1561 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
     1621 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
     1681 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1741 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1801 agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc
     1861 tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg
     1921 tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg
     1981 cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc
     2041 ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt
     2101 gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag
     2161 ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg
     2221 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
     2281 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
     2341 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
     2401 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
     2461 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
     2521 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
     2581 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     2641 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     2701 tggttattgt gctgtctcat cattttggca aagaattcag atctgcggcc gcctcgagaa
     2761 gcttgatcaa caagtttgta caaaaaaagc aggctagcgt cgacgccacc atgggctgca
     2821 tcaagagcaa gcgcaaggac aacctgaacg acgacggcgt ggactctaga atggtgagca
     2881 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2941 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     3001 ccctgaagct tctatgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     3061 ccctgggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact
     3121 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     3181 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     3241 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     3301 acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg
     3361 ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc gaccactacc
     3421 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct
     3481 accagtccgc cctgttcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     3541 tcctgaccgc cgccgggatc actgagggca tgaacgagct gtacctcgag gaaggttccc
     3601 aattctgggt aacggtgcag aggactgagg ccgccgagcg ctgtggcctg catggctcct
     3661 acgtgctgag ggtggaggct gaaaggctga ctctcctgac cgtgggggcc cagagtcaga
     3721 tactggagcc actcctgtcc tggccctaca ctctgttgcg tcgctatggc cgggacaagg
     3781 tcatgttctc tttcgaggcc ggccgccgct gcccctcagg ccctggaacc ttcaccttcc
     3841 agacggcaca gggaaatgac atcttccagg cagttgagac tgccatccac cggcagaagg
     3901 cccagggaaa ggccggaggt accagtgctg gtggtagtgc tggtggtagt gctggtggta
     3961 gtgctggtgg tagtgctggt ggttccggca gtgctggtgg tagtgctggt ggtagtacca
     4021 gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc tggtggtagt gctggtggtt
     4081 ccggcagtgc tggtggtagt gctggtggta gtaccagtgc tggtggtagt gctggtggta
     4141 gtgctggtgg tagtgctggt ggtagtgctg gtggttccgg cagtgctggt ggtagtgctg
     4201 gtggtagtac cagtgctggt ggtagtgctg gtggtagtgc tggtggtagt gctggtggta
     4261 gtgctggtgg ttccggaaaa ggagagttta agccgaatgt cgtgactaca gtagccagta
     4321 agtttattcc taatgaaggt acggactgga aagccaatgg cggccgcatg gtgagcaagg
     4381 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     4441 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     4501 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     4561 tgtcctgggg cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct
     4621 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     4681 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     4741 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     4801 actactttag cgacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca
     4861 acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgccgac cactaccagc
     4921 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     4981 agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     5041 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaatga gaattcaccc
     5101 agctttcttg tacaaagtgg ttgatcatcg atgatctaga gctcgctgat cagcctcgac
     5161 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     5221 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     5281 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     5341 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     5401 aacctgcagc ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5461 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5521 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5581 aaacctgtcg tgccagcgga tccattcatg aatgaattca tgtcgacata ctagttaaaa
     5641 gttttgttac tttatagaag aaattttgag tttttgtttt tttttaataa ataaataaac
     5701 ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata ataaaactta
     5761 atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca catgcgtcaa
     5821 ttttacgcat gattatcttt aacgtacgtc acaatatgat tatctttcta gggttaatct
     5881 agtatacgcg tatgcggccg cttaattaat ccagcttttg ttccctttag tgagggttaa
     5941 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     6001 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     6061 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6121 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6181 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     6241 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6301 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6361 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6421 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6481 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6541 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6601 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6661 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6721 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6781 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6841 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6901 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6961 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     7021 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     7081 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7141 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7201 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7261 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7321 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7381 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7441 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7501 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7561 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7621 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7681 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7741 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7801 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7861 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7921 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7981 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     8041 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     8101 gaaaagtgcc ac
//
