LOCUS       sequence-258388-        6757 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Research the effect of gene orientation of transcription.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6757)
  AUTHORS   Yeung E, Dy AJ, Martin KB, Ng AH, Del Vecchio D, Beck JL, Collins 
            JJ, Murray RM
  TITLE     Biophysical Constraints Arising from Compositional Context in 
            Synthetic Gene Networks.
  JOURNAL   Cell Syst. 2017 Jul 26;5(1):11-24.e12. doi: 
            10.1016/j.cels.2017.06.001. Epub 2017 Jul 19.
  PUBMED    28734826
REFERENCE   2  (bases 1 to 6757)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cels.2017.06.001"; journalName: "Cell Syst"; date: 
            "2017-07-26- 26"; volume: "5"; issue: "1"; pages: "11-24.e12"
COMMENT     Sequence Label: pEY31CBiT
FEATURES             Location/Qualifiers
     source          1..6757
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(215..236)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     564..583
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     823..844
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1116..1736)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     1644..1663
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    1838..1854
                     /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)."
     primer_bind     complement(2222..2240)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2308..2412
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2413..2481,2482..3273)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHATRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2631..2650)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3444..4032
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3933..3952
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4186..4203
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4399..4417
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4424..4442
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             4543..5622
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4562..4581)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5823..6500
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      6575..6661
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gggcatgact atcgtcgccg cacttatgac tgtgttcttt atcatgcaac tcgtaggaca
       61 ggtgccggca gcgctcttcc ggcttgcggc gacgatagtc atgccccgcg cccaccggaa
      121 ggagcgaaga caatgcccga cctcaagggc atcggtcgac acagaaaagc ccgcctttcg
      181 gcgggctttg ctcgagttag atcccggcgg cggtcacgaa ctccagcagg accatgtgat
      241 cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg
      301 gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca
      361 cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct
      421 tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga
      481 tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc
      541 cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc
      601 gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt
      661 cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg
      721 gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat
      781 cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga
      841 ccaggatggg aacaacgcca gtgaaaagct ccatgttcac attagaaagt ctcctgtgca
      901 tgattaagat gtttcagtac gaaaattgct ttcattgttg atctcctttt taagtgaact
      961 tgggcccaca ttaatatgtg aaagaatttg agggagatag aggggaatag gtgatgatag
     1021 agaggtgtgg ttaatgatgg aagaagttag attagtttgg aatgtatgag tcattttcat
     1081 taagctgcta aagcgtagtt ttcgtcgttt gctgcagacc cactttcaca tttaagttgt
     1141 ttttctaatc cgcatatgat caattcaagg ccgaataaga aggctggctc tgcaccttgg
     1201 tgatcaaata attcgatagc ttgtcgtaat aatggcggca tactatcagt agtaggtgtt
     1261 tccctttctt ctttagcgac ttgatgctct tgatcttcca atacgcaacc taaagtaaaa
     1321 tgcccaacag cgctgagtgc atataatgca ttctctagtg aaaaaccttg ttggcataaa
     1381 aaggctaatt gattttcgag agtttcatac tgtttttctg taggccgtgt acctaaatgt
     1441 acttttgctc catcgcgatg acttagtaaa gcacatctaa aacttttagc gttattacgt
     1501 aaaaaatctt gccagctttc cccttctaaa gggcaaaagt gagtatggtg cctatctaac
     1561 atctcaatgg ctaaggcgtc gagcaaagcc cgcttatttt ttacatgcca atacaatgta
     1621 ggctgctcta cacctagctt ctgggcgagt ttacgggttg ttaaaccttc gattccgacc
     1681 tcattaagca gctctaatgc gctgttaatc actttacttt tatctaatct agacattaga
     1741 aaccctcccc tgcatgatta agatgtttca gtacgaaaat tgctttcatt gttgatctcc
     1801 tttttaagtg aacttgggcc cgcttgcttt cagtatcttg ttatccgctc acaatgtcaa
     1861 tgttatccgc tcacatttat gaacagcaat gcgtggttgt cttcgttacg tccgtcacgt
     1921 tcacgcatca ggcggatatc gttagcccac ccagcaaaat tcggaacagc aatgcgtggt
     1981 tgtcttcgtt acgtccgtca cgttcacgca tcaggcggat atcgttagcc caccaagcaa
     2041 aattcggttt tctggctgat ggtgcgatag tcatcaccat gtcaaacatc cactctgcgg
     2101 cggtcaggtc atctgctgtc ccccacttgc tggcgctctg aattgcagca tccggtttca
     2161 ccacagaaag gtcgttttct ggctggtcag aggattcgcc agaattcttg aacacgaaag
     2221 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     2281 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2341 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2401 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2461 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2521 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2581 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     2641 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     2701 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     2761 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     2821 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     2881 gcaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     2941 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     3001 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3061 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3121 gagcgtgggt cacgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3181 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3241 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3301 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3361 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3421 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3481 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3541 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     3601 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3661 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3721 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3781 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3841 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3901 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3961 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4021 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4081 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4141 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4201 gaggaagcgg aagagcgctg ccggcacctg tcctacgagt tgcatgataa agaacacagt
     4261 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagcgaa gacaatgccc
     4321 gacgctgggt gggctaacga tatccgcctg atgcgtgaac gtgacggacg taacgaagac
     4381 aaccacgcat tgctgttctc cctatcagtg atagagattg acatccctat cagtgataga
     4441 gatactgaaa gcgaagcggg cccaagttca cttaaaaagg agatcaacaa tgaaagcaat
     4501 tttcgtactg aaacatctta atcatgcgga ggatcgtttc taatgaaacc agtaacgtta
     4561 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     4621 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     4681 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     4741 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     4801 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     4861 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     4921 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     4981 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaggacggt
     5041 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     5101 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     5161 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     5221 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     5281 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     5341 gatatctcgg tagtgggata cgacgatacc gaggacagct catgttatat cccgccgtta
     5401 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     5461 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     5521 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     5581 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agaatgactc atacattcca
     5641 aactaatcta acttcttcca tcattaacca cacctctcta tcatcaccta ttcccctcta
     5701 tctccctcaa attctttcac atattaatgt gggcccaagt tcacttaaaa aggagatcaa
     5761 caatgaaagc aattttcgta ctgaaacatc ttaatcatgc agaggagtct ttctaatgtg
     5821 aaatggcgag tagcgaagac gttatcaaag agttcatgcg tttcaaagtt cgtatggaag
     5881 gttccgttaa cggtcacgag ttcgaaatcg aaggtgaagg tgaaggtcgt ccgtacgaag
     5941 gtacccagac cgctaaactg aaagttacca aaggtggtcc gctgccgttc gcttgggaca
     6001 tcctgtcccc gcagttccag tacggttcca aagcttacgt taaacacccg gctgacatcc
     6061 cggactacct gaaactgtcc ttcccggaag gtttcaaatg ggaacgtgtt atgaacttcg
     6121 aagacggtgg tgttgttacc gttacccagg actcctccct gcaagacggt gagttcatct
     6181 acaaagttaa actgcgtggt accaacttcc cgtccgacgg tccggttatg cagaaaaaaa
     6241 ccatgggttg ggaagcttcc accgaacgta tgtacccgga agacggtgct ctgaaaggtg
     6301 aaatcaaaat gcgtctgaaa ctgaaagacg gtggtcacta cgacgctgaa gttaaaacca
     6361 cctacatggc taaaaaaccg gttcagctgc cgggtgctta caaaaccgac atcaaactgg
     6421 acatcacctc ccacaacgaa gactacacca tcgttgaaca gtacgaacgt gctgaaggtc
     6481 gtcactccac cggtgcttaa gcagcgaacg acgaaaatta cgcccttgca gcgtgaaagc
     6541 ttaattagct gatctagacg cgtgctagag gcatcaaata aaacgaaagg ctcagtcgaa
     6601 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     6661 tccgccgccc tagacctagg gcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6721 gggtcgggca ttgtcttcgc tccttccggt gggcgcg
//
