LOCUS       sequence-161598-        6703 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CRE and iRFP670 under two separate PGK promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6703)
  AUTHORS   Daneshvar K, Pondick JV, Kim BM, Zhou C, York SR, Macklin JA, 
            Abualteen A, Tan B, Sigova AA, Marcho C, Tremblay KD, Mager J, Choi 
            MY, Mullen AC
  TITLE     DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC 
            Expression to Control Definitive Endoderm Differentiation of 
            Embryonic Stem Cells.
  JOURNAL   Cell Rep. 2016 Oct 4;17(2):353-365. doi: 
            10.1016/j.celrep.2016.09.017.
  PUBMED    27705785
REFERENCE   2  (bases 1 to 6703)
  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.celrep.2016.09"; journalName: "Cell Rep"; date: 
            "2016-10-4- 4"; volume: "17"; issue: "2"; pages: "353-365"
COMMENT     Sequence Label: pCRE-iRFP670
FEATURES             Location/Qualifiers
     source          1..6703
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        56..160
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(161..229,230..1021)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(379..398)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1192..1780
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1681..1700
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1934..1951
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2068..2089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2104..2109,2110..2127,2128..2134)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2142..2158
                     /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     2147..2169
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2166..2182
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2166..2182
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2203..2221
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2250..2266
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2291..2790
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             2815..2835
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2836..3864
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     primer_bind     complement(3888..3904)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    3924..4148
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4226..4242
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      4778..4787
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4784..5719
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAG
                     AFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLH
                     RHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVM
                     LYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSR
                     IVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYE
                     PRAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     polyA_signal    5750..5974
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6065..6084)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6066..6084)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6094..6111)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6094..6110)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6103..6125)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6252..6680
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6339..6358)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6549..6570
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 atttatgcaa tcctactatg caatgtatcc aggtggcact tttcggggaa atgtgcgcgg
       61 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
      121 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
      181 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
      241 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
      301 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
      361 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
      421 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
      481 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
      541 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
      601 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
      661 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
      721 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
      781 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
      841 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
      901 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
      961 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     1021 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     1081 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     1141 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     1201 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     1261 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     1321 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     1381 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     1441 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     1501 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     1561 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1621 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1681 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1741 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1801 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     1861 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     1921 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     1981 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     2041 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     2101 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     2161 tcacacagga aacagctatg accatgatta cgccaagctc gaaattaacc ctcactaaag
     2221 ggaacaaaag ctggtaccgg gccccccctc gaggtcgacg gtatcgataa gcttgatatc
     2281 gaattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
     2341 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
     2401 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
     2461 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
     2521 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
     2581 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
     2641 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
     2701 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
     2761 ttctcctctt cctcatctcc gggcctttcg acctgcagct cgaggtcgac catgcccaag
     2821 aagaagagga aggtgtccaa tttactgacc gtacaccaaa atttgcctgc attaccggtc
     2881 gatgcaacga gtgatgaggt tcgcaagaac ctgatggaca tgttcaggga tcgccaggcg
     2941 ttttctgagc atacctggaa aatgcttctg tccgtttgcc ggtcgtgggc ggcatggtgc
     3001 aagttgaata accggaaatg gtttcccgca gaacctgaag atgttcgcga ttatcttcta
     3061 tatcttcagg cgcgcggtct ggcagtaaaa actatccagc aacatttggg ccagctaaac
     3121 atgcttcatc gtcggtccgg gctgccacga ccaagtgaca gcaatgctgt ttcactggtt
     3181 atgcggcgga tccgaaaaga aaacgttgat gccggtgaac gtgcaaaaca ggctctagcg
     3241 ttcgaacgca ctgatttcga ccaggttcgt tcactcatgg aaaatagcga tcgctgccag
     3301 gatatacgta atctggcatt tctggggatt gcttataaca ccctgttacg tatagccgaa
     3361 attgccagga tcagggttaa agatatctca cgtactgacg gtgggagaat gttaatccat
     3421 attggcagaa cgaaaacgct ggttagcacc gcaggtgtag agaaggcact tagcctgggg
     3481 gtaactaaac tggtcgagcg atggatttcc gtctctggtg tagctgatga tccgaataac
     3541 tacctgtttt gccgggtcag aaaaaatggt gttgccgcgc catctgccac cagccagcta
     3601 tcaactcgcg ccctggaagg gatttttgaa gcaactcatc gattgattta cggcgctaag
     3661 gatgactctg gtcagagata cctggcctgg tctggacaca gtgcccgtgt cggagccgcg
     3721 cgagatatgg cccgcgctgg agtttcaata ccggagatca tgcaagctgg tggctggacc
     3781 aatgtaaata ttgtcatgaa ctatatccgt aacctggata gtgaaacagg ggcaatggtg
     3841 cgcctgctgg aagatggcga ttagccatta acgcgtaaat gattgcagat ccactagttc
     3901 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3961 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4021 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4081 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     4141 ctctatggct tctgaggaaa gaaccagctg gggctcgaga tccactagtt ctagcctcga
     4201 ggctagagcg taccgggccc cccctcgagg tcgacggtat cgataagctt gatatcgaat
     4261 tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc
     4321 cgctggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg
     4381 tagcgccaac cggctccgtt ctttggtggc cccttcgcgc cacttctact cctcccctag
     4441 tcaggaagtt tcccccagca agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg
     4501 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
     4561 gggcagcggc caatagcagc tttgttcctt cgctttctgg gctcagaggc tgggaagggg
     4621 tgggtccggg ggcgggctca ggggcgggct caggggcggg cgggcgcccg aaggtcctcc
     4681 cgaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc
     4741 tcatctccgg gcctttcgac ctgcagatcc accggtcgcc accatggcgc gtaaggtcga
     4801 tctcacctcc tgcgatcgcg agccgatcca catccccggc agcattcagc cgtgcggctg
     4861 cctgctagcc tgcgacgcgc aggcggtgcg gatcacgcgc attacggaaa atgccggcgc
     4921 gttctttgga cgcgaaactc cgcgggtcgg tgagctactc gccgattact tcggcgagac
     4981 cgaagcccat gcgctgcgca acgcactggc gcagtcctcc gatccaaagc gaccggcgct
     5041 gatcttcggt tggcgcgacg gcctgaccgg ccgcaccttc gacatctcac tgcatcgcca
     5101 tgacggtaca tcgatcatcg agttcgagcc tgcggcggcc gaacaggccg acaatccgct
     5161 gcggctgacg cggcagatca tcgcgcgcac caaagaactg aagtcgctcg aagagatggc
     5221 cgcacgggtg ccgcgctatc tgcaggcgat gctcggctat caccgcgtga tgttgtaccg
     5281 cttcgcggac gacggctccg ggatggtgat cggcgaggcg aagcgcagcg acctcgagag
     5341 ctttctcggt cagcactttc cggcgtcgct ggtcccgcag caggcgcggc tactgtactt
     5401 gaagaacgcg atccgcgtgg tctcggattc gcgcggcatc agcagccgga tcgtgcccga
     5461 gcacgacgcc tccggcgccg cgctcgatct gtcgttcgcg cacctgcgca gcatctcgcc
     5521 ctgccatctc gaatttctgc ggaacatggg cgtcagcgcc tcgatgtcgc tgtcgatcat
     5581 cattgacggc acgctatggg gattgatcat ctgtcatcat tacgagccgc gtgccgtgcc
     5641 gatggcgcag cgcgtcgcgg ccgaaatgtt cgccgacttc ttatcgctgc acttcaccgc
     5701 cgcccaccac caacgctaag cggccctaga gctcgctgat cagcctcgac tgtgccttct
     5761 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     5821 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     5881 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     5941 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggaaagaac cagctggggc
     6001 tcgagatcca ctagttctag cctcgaggct agagcggccg ccaccgcggt ggagctccaa
     6061 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     6121 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     6181 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     6241 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     6301 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     6361 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     6421 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     6481 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     6541 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     6601 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     6661 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt aca
//
