LOCUS       Exported                8311 bp ds-DNA     circular SYN 25-DEC-2020
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8311)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Dec 25, 2020 from SnapGene 5.1.7
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8311
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..2848
                     /label=Backbone
                     /note="color: #ffcc99"
     protein_bind    11..27
                     /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)."
                     /note="color: #31849b"
     promoter        complement(35..65)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 35 .. 41 / #ffffff / -10
                        2: 42 .. 59 / #ffffff
                        3: 60 .. 65 / #ffffff / -35"
     protein_bind    80..101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     rep_origin      complement(389..974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(1145..2005)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 1145 .. 1936 / #ccffcc
                        2: 1937 .. 2005 / #ccffcc / signal sequence
                     Cleavage site after base 1936"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2006..2110)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
     rep_origin      2137..2592
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     primer_bind     2732..2749
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     misc_feature    2852..3551
                     /label=5' homology arm
                     /note="color: #3366ff"
     misc_feature    2864..2883
                     /label=Primer 610
                     /note="color: #a6acb3; direction: RIGHT"
     promoter        3565..4743
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    complement(3677..3697)
                     /label=Primer 764
                     /note="color: #a6acb3; direction: LEFT"
     intron          3796..4734
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
                     /note="color: #------"
     regulatory      4757..4766
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     CDS             4763..5482
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="This feature has 3 segments:
                        1: 4763 .. 4765 / #05fd14
                        2: 4766 .. 4768 / #05fd14 / 1a
                        3: 4769 .. 5482 / #05fd14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    4823..4843
                     /label=Primer 225
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    complement(5389..5409)
                     /label=Primer 226
                     /note="color: #a6acb3; direction: LEFT"
     polyA_site      5634..5689
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
                     /note="color: #a6acb3"
     protein_bind    5749..5782
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
                     /note="color: #31849b; direction: RIGHT"
     promoter        5819..6315
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    complement(5907..5926)
                     /label=Primer 665
                     /note="color: #a6acb3; direction: LEFT"
     CDS             6355..6954
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /note="color: #ccffcc"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    7032..7484
                     /label=PGK polyA
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    7400..7421
                     /label=Primer 666
                     /note="color: #a6acb3; direction: RIGHT"
     protein_bind    7535..7568
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
                     /note="color: #31849b; direction: RIGHT"
     misc_feature    7604..8306
                     /label=3' homology arm
                     /note="color: #3366ff"
     misc_feature    complement(7960..7979)
                     /label=Primer 611
                     /note="color: #a6acb3; direction: LEFT"
ORIGIN
        1 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
       61 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
      121 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
      181 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
      241 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
      301 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
      361 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
      421 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
      481 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
      541 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
      601 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
      661 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
      721 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
      781 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
      841 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
      901 gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      961 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1021 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1081 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1141 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1201 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1261 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     1321 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1381 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1441 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1501 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1561 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1621 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1681 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1741 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1801 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1861 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     1921 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     1981 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2041 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2101 gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa
     2161 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2221 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2281 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2341 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2401 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2461 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2521 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2581 cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     2641 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     2701 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg ccaagctgat
     2761 ctatacattg aatcaatatt ggcaattagc catattagtc attggttata tagcataaat
     2821 caatattggc tattggccat tgcatacgtt tcttgtgaac tcatctcgtg cctgcaagac
     2881 atcaaaagtt tctccaaaga catcccttcc tctctgccct catcctatat caaggtctta
     2941 gctccttgaa gactgcatta atgtgtcttt tgccttcatc ccccccccac cctcccgctt
     3001 cctctagtcc ttgtgtttac caaaatactt ttgctaaaac ctgtatgtct agcttctgct
     3061 gatactctta gcaatactcc cttatttcct tctggcgcca tttgccaatc accagctaat
     3121 ggctttgctc tttgcatggt acctgtttct gtcctactag cttctagtct gaaaaggaag
     3181 cctttactag cagcttcaac ccagtcctcc ttaccttgca cctattaacg ttggttcaac
     3241 atgaagggga agtaagataa gtggaagagg atacacagga aaggtggaac tggattcagg
     3301 tgtctgtgtt ctgccattga cacttgggac tgcgttccag cttccacctg tggaaaccag
     3361 aaagcatttg ccaaaaattc taaacagagg gtctatttct gaagctgagg aatcacatgg
     3421 agtgaatagc atgggggaag gggattacta atgaggctga aagaaaagta ccaggaagag
     3481 actgacttca tgcataatcc ataaagccag gttttcttga aaggtgacct gctttcttgt
     3541 attcctgcaa gaaacggctc gaggggctcc ggtgcccgtc agtgggcaga gcgcacatcg
     3601 cccacagtcc ccgagaagtt gtggggaggg gtcggcaatt gaagcggtgc ctagagaagg
     3661 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     3721 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     3781 gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt
     3841 tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc
     3901 gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg
     3961 cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg
     4021 caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga
     4081 cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac
     4141 actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca
     4201 tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa
     4261 gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg
     4321 gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct
     4381 gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc
     4441 acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac
     4501 cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt
     4561 tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt
     4621 aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct
     4681 tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg
     4741 tgaggggatc gtcgacgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     4801 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     4861 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     4921 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     4981 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     5041 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     5101 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     5161 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     5221 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     5281 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     5341 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     5401 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     5461 catggacgag ctgtacaagt aaagcggccg cactcctcag gtgcaggctg cctatcagaa
     5521 ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc tttttccctc
     5581 tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag
     5641 gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac
     5701 atatgggagg gcgatcgcca gtactagtga acctcttcga gggacctaat aacttcgtat
     5761 agcatacatt atacgaagtt atattaaggg ttccggatct cgacctcgaa attctaccgg
     5821 gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctggca
     5881 cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc ggtagcgcca
     5941 accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa
     6001 gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct
     6061 cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg
     6121 cagcggccaa tagcagcttt gctccttcgc tttctgggct cagcagctgg gaagggtggg
     6181 tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga
     6241 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca
     6301 tctccgggcc tttcgacctg catccatcta gatctcgagc agctgaagct taccatgacc
     6361 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     6421 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     6481 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     6541 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     6601 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     6661 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     6721 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     6781 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     6841 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     6901 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg
     6961 ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc gatgatatca
     7021 gatccccggg atgcagaaat tgatgatcta ttaaacaata aagatgtcca ctaaaatgga
     7081 agtttttcct gtcatacttt gttaagaagg gtgagaacag agtacctaca ttttgaatgg
     7141 aaggattgga gctacggggg tgggggtggg gtgggattag ataaatgcct gctctttact
     7201 gaaggctctt tactattgct ttatgataat gtttcatagt tggatatcat aatttaaaca
     7261 agcaaaacca aattaagggc cagctcattc ctcccactca tgatctatag atctatagat
     7321 ctctcgtggg atcattgttt ttctcttgat tcccactttg tggttctaag tactgtggtt
     7381 tccaaatgtg tcagtttcat agcctgaaga acgagatcag cagcctctgt tccacataca
     7441 cttcattctc agtattgttt tgccaagttc taattccatc agaagctggt cgagatccta
     7501 agcttggctg gacgtaaact cctcttcaga cctaataact tcgtatagca tacattatac
     7561 gaagttatat taagggttat tgaatatgat cggaattgcg gccctgacct gttggggtct
     7621 ttaaagctca aggaaaaagg ccatagttga tttctcctaa atcaagatag agtccaatta
     7681 actttttttt ttttttaaga tggagtctca ctctgtcgcc aggctggact gcagtggcgt
     7741 gatctcagct cactgcaacc tctgcctccc aggttcaggc gattcttctg tctcagcctc
     7801 ccgagtagct gggactacaa gtgtgggcca ccatgcccag ctagttttgt atttttagta
     7861 gagatgggat ttcaccatct tggctaggat ggtcttgatc tcttgacctt gtgattcgcc
     7921 tgcctctgcc tcccagagtg ctgagattac aggcgtgagc cacagtgccc aatttccatg
     7981 ggttttcaag aaaaacttaa cttacttaaa cttcagatca cttccttaaa ggaacatact
     8041 gagcatagct tggctggtta gaaattagga agactagctt gggctacatg gtaagaccct
     8101 gcctctacaa aaaataagaa aaaagttagt ggcatgtgcc tgtagttcca tctacttggg
     8161 aggctgaggt gagaggatcg cttgagccca ggaggttgaa gctgcagtga gccacgattg
     8221 caccactgta ctccagcctg ggtgacacag agtgagaccc tgtctccaaa caaaattagg
     8281 aagggtttca gagaggaaat aaacacggcc g
//
