LOCUS       Exported                6747 bp ds-DNA     circular SYN 23-NOV-2023
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6747)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, May 21, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6747
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        26..404
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
                     /note="color: #ffffff; direction: RIGHT"
                     /note="color: #00ff00"
     protein_bind    34..52
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
                     /note="color: #ffc0cb"
     protein_bind    70..88
                     /gene="tetO"
                     /label=tet operator
                     /label=tet operator(1)
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
                     /note="color: #ffc0cb"
     protein_bind    106..124
                     /gene="tetO"
                     /label=tet operator
                     /label=tet operator(2)
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
                     /note="color: #ffc0cb"
     protein_bind    142..160
                     /gene="tetO"
                     /label=tet operator
                     /label=tet operator(3)
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
                     /note="color: #ffc0cb"
     protein_bind    178..196
                     /gene="tetO"
                     /label=tet operator
                     /label=tet operator(4)
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
                     /note="color: #ffc0cb"
     protein_bind    214..232
                     /gene="tetO"
                     /label=tet operator
                     /label=tet operator(5)
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
                     /note="color: #ffc0cb"
     protein_bind    250..268
                     /gene="tetO"
                     /label=tet operator
                     /label=tet operator(6)
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="color: #31849b"
                     /note="color: #ffc0cb"
     primer_bind     313..337
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
                     /note="color: black; sequence: AGCTCGTTTAGTGAACCGTCAGATC"
                     /note="color: #ffc0cb; direction: RIGHT"
     promoter        405..423
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             438..2087
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /note="color: #ffcc00"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    2138..2362
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      2408..2836
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        2850..3179
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
                     /note="color: #ffffff; direction: RIGHT"
     rep_origin      3030..3165
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     CDS             3246..4040
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /note="color: #ccffcc"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    4214..4335
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(4384..4400)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    4408..4424
                     /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(4432..4462)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 4432 .. 4438 / #ffffff / -10
                        2: 4439 .. 4456 / #ffffff
                        3: 4457 .. 4462 / #ffffff / -35"
     protein_bind    4477..4498
                     /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(4786..5371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(5542..6402)
                     /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: 5542 .. 6333 / #ccffcc
                        2: 6334 .. 6402 / #ccffcc / signal sequence
                     Cleavage site after base 6333"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6403..6507)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 gatgtacggg ccagatatac gcgttgagtt tactccctat cagtgataga gaacgtatga
       61 agagtttact ccctatcagt gatagagaac gtatgcagac tttactccct atcagtgata
      121 gagaacgtat aaggagttta ctccctatca gtgatagaga acgtatgacc agtttactcc
      181 ctatcagtga tagagaacgt atctacagtt tactccctat cagtgataga gaacgtatat
      241 ccagtttact ccctatcagt gatagagaac gtataagctt taggcgtgta cggtgggcgc
      301 ctataaaagc agagctcgtt tagtgaaccg tcagatcgcc tggagcaatt ccacaacact
      361 tttgtcttat accaactttc cgtaccactt cctaccctcg taaataatac gactcactat
      421 agggagaccc aagctggatg gaagatgcca aaaacattaa gaagggccca gcgccattct
      481 acccactcga agacgggacc gccggcgagc agctgcacaa agccatgaag cgctacgccc
      541 tggtgcccgg caccatcgcc tttaccgacg cacatatcga ggtggacatt acctacgccg
      601 agtacttcga gatgagcgtt cggctggcag aagctatgaa gcgctatggg ctgaatacaa
      661 accatcggat cgtggtgtgc agcgagaata gcttgcagtt cttcatgccc gtgttgggtg
      721 ccctgttcat cggtgtggct gtggccccag ctaacgacat ctacaacgag cgcgagctgc
      781 tgaacagcat gggcatcagc cagcccaccg tcgtattcgt gagcaagaaa gggctgcaaa
      841 agatcctcaa cgtgcaaaag aagctaccga tcatacaaaa gatcatcatc atggatagca
      901 agaccgacta ccagggcttc caaagcatgt acaccttcgt gacttcccat ttgccacccg
      961 gcttcaacga gtacgacttc gtgcccgaga gcttcgaccg ggacaaaacc atcgccctga
     1021 tcatgaacag tagtggcagt accggattgc ccaagggcgt agccctaccg caccgcaccg
     1081 cttgtgtccg attcagtcat gcccgcgacc ccatcttcgg caaccagatc atccccgaca
     1141 ccgctatcct cagcgtggtg ccatttcacc acggcttcgg catgttcacc acgctgggct
     1201 acttgatctg cggctttcgg gtcgtgctca tgtaccgctt cgaggaggag ctattcttgc
     1261 gcagcttgca agactataag attcaatctg ccctgctggt gcccacacta tttagcttct
     1321 tcgctaagag cactctcatc gacaagtacg acctaagcaa cttgcacgag atcgccagcg
     1381 gcggggcgcc gctcagcaag gaggtaggtg aggccgtggc caaacgcttc cacctaccag
     1441 gcatccgcca gggctacggc ctgacagaaa caaccagcgc cattctgatc acccccgaag
     1501 gggacgacaa gcctggcgca gtaggcaagg tggtgccctt cttcgaggct aaggtggtgg
     1561 acttggacac cggtaagaca ctgggtgtga accagcgcgg cgagctgtgc gtccgtggcc
     1621 ccatgatcat gagcggctac gttaacaacc ccgaggctac aaacgctctc atcgacaagg
     1681 acggctggct gcacagcggc gacatcgcct actgggacga ggacgagcac ttcttcatcg
     1741 tggaccggct gaagagcctg atcaaataca agggctacca ggtagcccca gccgaactgg
     1801 agagcatcct gctgcaacac cccaacatct tcgacgccgg ggtcgccggc ctgcccgacg
     1861 acgatgccgg cgagctgccc gccgcagtcg tcgtgctgga acacggtaaa accatgaccg
     1921 agaaggagat cgtggactat gtggccagcc aggttacaac cgccaagaag ctgcgcggtg
     1981 gtgttgtgtt cgtggacgag gtgcctaaag gactgaccgg caagttggac gcccgcaaga
     2041 tccgcgagat tctcattaag gccaagaagg gcggcaagat cgccgtgtcg actgtttaag
     2101 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     2161 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2221 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2281 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2341 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     2401 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2461 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2521 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2581 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2641 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2701 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2761 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2821 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     2881 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     2941 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3001 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3061 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     3121 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3181 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     3241 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3301 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3361 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3421 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3481 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3541 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     3601 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     3661 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     3721 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     3781 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     3841 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     3901 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     3961 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4021 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4081 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4141 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4201 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4261 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4321 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     4381 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4441 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4501 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4561 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4621 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4681 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4741 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4801 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4861 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4921 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4981 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5041 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5101 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5161 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5221 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5281 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtttttttg tttgcaagca
     5341 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5401 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5461 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5521 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5581 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5641 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5701 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5761 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5821 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5881 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5941 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6001 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6061 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6121 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6181 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6241 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6301 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6361 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6421 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6481 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     6541 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     6601 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     6661 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     6721 agggttaggc gttttgcgct gcttcgc
//
