LOCUS       CK539-PB-EF1a-MG        5883 bp    DNA     circular UNA 17-MAR-2021
DEFINITION  Gibson/Homology Cloning ligation product.
ACCESSION   urn.local...d-dr5t6q8
VERSION     urn.local...d-dr5t6q8
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     primer_bind     complement(29..51)
                     /Tm=66
                     /Description="pGEX vectors, reverse primer"
                     /label="pGEX 3'"
     primer_bind     151..170
                     /Tm=54
                     /Description="pRS vectors, use to sequence yeast
                     selectable marker"
                     /label="pRS-marker"
     primer_bind     364..386
                     /Tm=61
                     /Description="In lacZ gene"
                     /label="M13/pUC Forward"
     primer_bind     378..395
                     /Tm=56
                     /Description="In lacZ gene. Also called M13-F20 or M13
                     (-21) Forward"
                     /label="M13 Forward"
     primer_bind     379..395
                     /note="<html><body>common sequencing primer, one of
                     multiple similar variants</body></html>"
                     /label="M13 fwd"
     repeat_region   418..480
                     /note="<html><body><!--StartFragment-->piggyBac
                     transposon-specific inverted terminal repeat sequence
                     (ITR)<!--EndFragment--></body></html>"
                     /label="piggyBac right (3') inverted repeat"
     promoter        1424..2605
                     /note="<html><body>strong constitutive promoter for human
                     elongation factor EF-1α</body></html>"
                     /label="EF-1α promoter"
     intron          1654..2596
                     /note="<html><body>intron upstream of the start codon of
                     human EF-1α</body></html>"
                     /label="EF-1α intron A"
     primer_bind     2553..2573
                     /Tm=57
                     /Description="Human elongation factor-1a promoter, forward
                     primer"
                     /label="EF1a-F"
     primer_bind     2607..2631
                     /Tm=60
                     /Description="Human CMV promoter, forward primer"
                     /label="LNCX"
     protein_bind    2677..2801
                     /bound_moiety="<html><body>LR Clonase™</body></html>"
                     /gene="<html><body>mutant version
                     of&nbsp;<i>att</i>R</body></html>"
                     /note="<html><body>recombination site for the Gateway® LR
                     reaction</body></html>"
                     /label="attR1"
     primer_bind     2677..2702
                     /%GC="34.6"
                     /Hairpin_Tm="40.2"
                     /Self_Dimer_Tm="22.1"
                     /Tm="59.9"
                     /Pair_Dimer_Tm="9.4"
                     /Sequence="ACAAGTTTGTACAAAAAAGCTGAACG"
                     /created_by="primer3"
                     /Product_Size=654
                     /annotation_group="14978565151285e0c5e822-04f6-4503-91aa-8
                     d4ff07d5badMegaGate-PolyA Cassette"
                     /#_Off-target_Sites=0
                     /Off-target_Template="MegaGate-PolyA Cassette"
                     /Extension="GACCTCCATAGAAGACAC"
                     /label="MegaGate-PolyA Cassette (1 F) - 3_PB-EF1a-TetOn3G
                     (digested by ScaI, BsaI) Fragment 2 -
                     AmpR_BsaI_Cleanup_gBlock (digested by SphI, SapI) Fragment
                     2 - SapI_CleanUp_gBlock (digested by BbsI, EcoRV) Fragment
                     2"
     motif           2817..2834
                     /created_by="wyssuser"
                     /label="I-SceI"
     CDS             2835..2879
                     /created_by="wyssuser"
                     /modified_by="wyssuser"
                     /label="Trademark"
     motif           complement(2880..2906)
                     /created_by="wyssuser"
                     /label="I-CeuI"
     protein_bind    complement(2922..3046)
                     /bound_moiety="<html><body>LR Clonase™</body></html>"
                     /gene="<html><body>mutant version of
                     <i>att</i>R</body></html>"
                     /note="<html><body>recombination site for the Gateway® LR
                     reaction</body></html>"
                     /label="attR2"
     CDS             3048..3074
                     /created_by="wyssuser"
                     /label="HA Tag"
     terminator      3075..3083
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /locus_tag="source:synthetic DNA construct(4)"
                     /ApEinfo_label="source:synthetic DNA construct"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /modified_by="wyssuser"
                     /label="STOPS"
     motif           3115..3133
                     /created_by="wyssuser"
                     /label="BsaI_BC_Cloning"
     motif           3162..3186
                     /created_by="wyssuser"
                     /modified_by="wyssuser"
                     /label="FP NotI_BgIII cloning"
     polyA_signal    3196..3330
                     /note="SV40 polyadenylation signal"
                     /locus_tag="SV40 poly(A) signal"
                     /ApEinfo_label="SV40 poly(A) signal"
                     /ApEinfo_fwdcolor="#ff3eee"
                     /ApEinfo_revcolor="#ff3eee"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /modified_by="wyssuser"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(3304..3330)
                     /%GC="29.6"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="58.5"
                     /Pair_Dimer_Tm="9.4"
                     /Sequence="AACTTGTTTATTGCAGCTTATAATGGT"
                     /created_by="primer3"
                     /Product_Size=654
                     /annotation_group="149795741373748dafd205-0cc8-430d-9144-f
                     f8702861eb8MegaGate-PolyA Cassette"
                     /#_Off-target_Sites=0
                     /Off-target_Template="MegaGate-PolyA Cassette"
                     /Extension="TTATTATATATATATTTTCTTGTTATAGAT"
                     /label="MegaGate-PolyA Cassette (654 R) -
                     3_PB-EF1a-TetOn3G (digested by ScaI, BsaI) Fragment 2 -
                     AmpR_BsaI_Cleanup_gBlock (digested by SphI, SapI) Fragment
                     2 - SapI_CleanUp_gBlock (digested by BbsI, EcoRV) Fragment
                     2"
     repeat_region   3602..3636
                     /note="<html><body>piggyBac transposon-specific inverted
                     terminal repeat sequence (ITR)</body></html>"
                     /label="piggyBac left (5') inverted repeat"
     primer_bind     complement(3662..3678)
                     /Tm=49
                     /Description="In lacZ gene. Also called M13-rev"
                     /label="M13 Reverse"
     primer_bind     complement(3662..3678)
                     /note="<html><body>common sequencing primer, one of
                     multiple similar variants</body></html>"
                     /label="M13 rev"
     primer_bind     complement(3675..3697)
                     /Tm=58
                     /Description="In lacZ gene"
                     /label="M13/pUC Reverse"
     protein_bind    3686..3702
                     /note="<html><body>The <i>lac</i> repressor binds to the
                     <i>lac</i> operator to inhibit transcription in <i>E.
                     coli</i>. This inhibition can be relieved by adding
                     lactose or isopropyl-β-D-thiogalactopyranoside
                     (IPTG).</body></html>"
                     /bound_moiety="<html><body><i>lac</i> repressor encoded by
                     <i>lacI</i></body></html>"
                     /label="lac operator"
     promoter        join(complement(3735..3740),complement(3717..3734),
                     complement(3710..3716))
                     /note="<html><body>promoter for the <i>E. coli lac</i>
                     operon</body></html>"
                     /label="lac promoter"
     protein_bind    3755..3776
                     /note="<html><body>CAP binding activates transcription in
                     the presence of cAMP.</body></html>"
                     /bound_moiety="<html><body><i>E. coli</i> catabolite
                     activator protein</body></html>"
                     /label="CAP binding site"
     primer_bind     complement(3893..3910)
                     /Tm=58
                     /Description="L4440 vector, forward primer"
                     /label="L4440"
     rep_origin      complement(4064..4652)
                     /direction="LEFT"
                     /note="<html><body>high-copy-number ColE1/pMB1/pBR322/pUC
                     origin of replication</body></html>"
                     /label="ori"
     primer_bind     complement(4144..4163)
                     /Tm=56
                     /Description="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
     CDS             complement(4823..5683)
                     /created_by="wyssuser"
                     /label="AmpR"
     primer_bind     5446..5465
                     /Tm=58
                     /Description="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     promoter        complement(5684..5788)
                     /gene="<html><body><i>bla</i></body></html>"
                     /label="AmpR promoter"
     primer_bind     5856..5874
                     /Tm=57
                     /Description="pBR322 vectors, upsteam of EcoRI site,
                     forward primer"
                     /label="pBRforEco"
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 caagctttat tatttaaccc
      421 tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg
      481 tgttttatcg gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat
      541 tatatttaca cttacatact aataataaat tcaacaaaca atttatttat gtttatttat
      601 ttattaaaaa aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttaaacat
      661 tctctctttt acaaaaataa acttattttg tactttaaaa acagtcatgt tgtaatcgat
      721 aataagtaat tagcttaacc tatacataat agaaacaaat tatacttatt agtcagtcag
      781 aaacaacttt ggcacatatc aatattatgc tctcgttaat taactcgagg gatccggcgc
      841 gcctgtcatt ctaaatctct ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt
      901 ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc
      961 cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg
     1021 agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc
     1081 tgggggcttt gggagggggc tgtccccgtg agctcccagg cgcgcctgtc attctaaatc
     1141 tctctttcag cctaaagctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc
     1201 agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc
     1261 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg
     1321 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg
     1381 ggctgtcccc gtgagctccc aggcgcgccg ctagcttcgt gaggctccgg tgcccgtcag
     1441 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     1501 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     1561 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     1621 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg
     1681 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct
     1741 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
     1801 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg
     1861 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
     1921 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
     1981 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
     2041 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
     2101 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
     2161 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
     2221 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
     2281 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
     2341 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
     2401 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
     2461 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
     2521 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
     2581 ttttcttcca tttcaggtgt cgtgagagct cgtttagtga accgtcagat cgcctggaga
     2641 cgccatccac gctgttttga cctccataga agacacacaa gtttgtacaa aaaagctgaa
     2701 cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag
     2761 actacataat actgtaaaac acaacatatc cagtcactat ggcgacagaa gaagtatagg
     2821 gataacaggg taattgttgt aagcgcgcta tgatggaggc tatgccacta gaatctgcgt
     2881 tcgctacctt aggaccgtta tagttagaag gaaagctcca tcatagtgac tggatatgtt
     2941 gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat
     3001 ttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggtatac ccatacgatg
     3061 ttccagatta cgcttagtaa tgagaaatac tcagaagatg tcactcacca tagagagacc
     3121 attaatgggt ctcctaggcc actgagcaag aactatgaat gagatcttta gacatattgc
     3181 ggccgctttc tccgcgatcc agacatgata agatacattg atgagtttgg acaaaccaaa
     3241 actagaatgc agtgaaaaaa atgccttatt tgtgaaattt gtgatgctat tgccttattt
     3301 gtaaccatta taagctgcaa taaacaagtt atctataaca agaaaatata tatataataa
     3361 gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa
     3421 agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc
     3481 agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg
     3541 tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat
     3601 gcatgcgtca attttacgca gactatcttt ctagggttaa aaaaagcttg gcgtaatcat
     3661 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3721 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3781 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3841 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg cacttccgct tcctcgctca
     3901 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3961 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4021 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4081 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4141 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4201 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4261 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4321 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4381 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4441 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4501 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4561 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4621 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4681 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4741 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4801 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4861 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4921 gggagggctt accatctggc cccagtgctg caatgatacc gcgtgaccca cgctcaccgg
     4981 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5041 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5101 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5161 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5221 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5281 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5341 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5401 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5461 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5521 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5581 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5641 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5701 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5761 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     5821 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     5881 gtc
//
