LOCUS       pBR_attB(bxb)_cc        5240 bp DNA     circular SYN 01-FEB-2023
DEFINITION  Clone 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 5240)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Feb 1, 2023 from SnapGene 6.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5240
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                        1: 143 .. 148 / #ffffff / -35
                        2: 149 .. 166 / #ffffff
                        3: 167 .. 173 / #ffffff / -10"
     protein_bind    181..197
                     /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"
     primer_bind     186..209
                     /label=M13rev(-48) (#12 & #177)
                     /note="color: black; sequence: AGCGGATAACAATTTCACACAGGA"
     promoter        253..463
                     /label=SV40 promoter
                     /note="SV40 early promoter"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        559..1736
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     intron          789..1727
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
                     /note="color: #------"
     primer_bind     1530..1552
                     /label=EF1a_qPCRfwd (#195)
                     /note="color: black; sequence: TTTTGGAGTACGTCGTCTTTAGG"
     primer_bind     complement(1621..1643)
                     /label=EF1a_qPCRrev (#196)
                     /note="color: black; sequence: GGAGAATTACATCAAGTGCCAAG"
     misc_recomb     1754..1795
                     /label=attB
                     /note="from myobacteriumphage bxb1 (Thomson et al 2012)"
                     /note="color: #33cccc; direction: RIGHT"
     protein_bind    1802..1835
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
                     /note="color: #31849b; direction: RIGHT"
     primer_bind     1835..1892
                     /label=CG5_BpiI_HiFi_Fwd
                     /note="color: black; sequence: 
                     TcatgatattcggcaagcaggcatcgACTAGTTAATTAAGATCGtagctgccTAgtct;
                      added: 2022-09-05"
     primer_bind     complement(1836..1860)
                     /label=neo4 (#13)
                     /note="color: black; sequence: CGATGCCTGCTTGCCGAATATCATG"
     misc_feature    1883..1886
                     /label=TGCC
                     /note="color: #3366ff"
     CDS             1918..2223
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /note="color: #993366"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     Region          2224..2261
                     /label=terminator
                     /note="color: #cc99b2"
     primer_bind     complement(2271..2290)
                     /label=pACYC-F (#RD237)
                     /note="p15A origin, forward primer"
                     /note="color: black; sequence: TGAAGTCAGCCCCATACGAT"
     primer_bind     complement(2288..2345)
                     /label=CG5_BpiI_HiFi_Rev
                     /note="color: black; sequence: 
                     CTCCTATACGAAGTTATcgtgaggaagagttcttgcagcGtcctcccatgtcttcTGA;
                      added: 2022-09-05"
     misc_feature    2299..2302
                     /label=GGGA
                     /note="color: #ff0000"
     primer_bind     2307..2328
                     /label=Puro_153fwd (#258)
                     /note="color: black; sequence: GCTGCAAGAACTCTTCCTCACG"
     protein_bind    2329..2361
                     /label=lox251
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
                     /note="color: #31849b; direction: RIGHT"
     primer_bind     complement(2369..2386)
                     /label=M13fwd (#10 & #176)
                     /note="color: black; sequence: TGTAAAACGACGGCCAGT"
     primer_bind     complement(2376..2397)
                     /label=LacZ_rev (#328)
                     /note="color: black; sequence: AGTCACGACGTTGTAAAACGAC"
     primer_bind     complement(2467..2490)
                     /label=pBSKrev (#18)
                     /note="color: black; sequence: GGCGATCGGTGCGGGCCTCTTCGC"
     rep_origin      2598..3053
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        3335..3439
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             3440..4300
                     /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: 3440 .. 3508 / #ccffcc / signal sequence
                        2: 3509 .. 4300 / #ccffcc
                     Cleavage site after base 3508"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3701..3720)
                     /label=Amp_seq3 (#119)
                     /note="color: black; sequence: TGTATGCGGCGACCGAGTTG"
     primer_bind     complement(4301..4320)
                     /label=Amp_seq1 (#120)
                     /note="color: black; sequence: GAGTAAACTTGGTCTGACAG"
     rep_origin      4471..5059
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     5155..5178
                     /label=ColE1_rev (#118)
                     /note="color: black; sequence: CCGTATTACCGCCTTTGAGTGAGC"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctta
      241 attccctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
      301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      361 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctttgc
      481 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct
      541 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
      601 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
      661 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
      721 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
      781 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc
      841 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc
      901 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg
      961 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc
     1021 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg
     1081 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta
     1141 tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg
     1201 cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc
     1261 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc
     1321 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag
     1381 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa
     1441 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc
     1501 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg
     1561 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag
     1621 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca
     1681 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa
     1741 ttcgccacca tggccggctt gtcgacgacg gcggtctccg tcgtcaggat catccggatc
     1801 cataacttcg tatagcatac attatacgaa gttatcatga tattcggcaa gcaggcatcg
     1861 actagttaat taagatcgta gctgcctagt cttcacacat aaccaggagg tcagattatg
     1921 cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag
     1981 agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg
     2041 ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg
     2101 cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct
     2161 gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata
     2221 taacccagcc cgcctaatga gcgggctttt ttttgaacaa aacaacttat atcgtatggg
     2281 gctgacttca gaagacatgg gaggacgctg caagaactct tcctcacgat aacttcgtat
     2341 aggagacttt atacgaagtt aagcgctcac tggccgtcgt tttacaacgt cgtgactggg
     2401 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     2461 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     2521 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     2581 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2641 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2701 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2761 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     2821 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2881 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
     2941 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3001 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     3061 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     3121 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3181 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3241 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     3301 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3361 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3421 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3481 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3541 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3601 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3661 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3721 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3781 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3841 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3901 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3961 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     4021 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     4081 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     4141 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     4201 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4261 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4321 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4381 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4441 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4501 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4561 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     4621 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4681 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4741 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4801 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4861 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4921 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4981 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5041 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5101 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     5161 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     5221 agtgagcgag gaagcggaag
//
